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The weak have no place with Aku.
~ The High Priestess
The Samurai is our mortal enemy. He must die at any cost.
~ The High Priestess' last words before she falls off the cliff by Ashi.
The High Priestess is the secondary antagonist of the fifth and final season of Samurai Jack. She is the nameless masked leader of the Cult of Aku as well as the mother and mistress of the Daughters of Aku and lives to serve her god Aku.
She was voiced by Grey DeLisle, who also played Vicky in the Fairy Odd Parents, Azula in Avatar: The Last Airbender, Duchess in Foster's Home for Imaginary Friends, Asajj Ventress in Genndy Tartakovsky's Star Wars: Clone Wars, Lola Loud in The Loud House and Jubileus in Bayonetta.
The appearance of the High Priestess was never fully seen, but a silhouette can be discerned when she is giving birth to her daughters during a ceremony. She was briefly seen in the back when she was getting dressed into her outfit - she appears to be slim and has long black hair. Like the other cultists, she wears a long black robe, with a pink female mask and a head covering with six Aku-like horns -- unlike the usual four that regular cult members have (this could possibly signify her role as the leader of the cult).
Judging from silhouettes and taking into consideration how Ashi and her sisters do not resemble Aku (who's revealed to be their biological father in the second-to-last episode of the final season), the High Priestess seems to resemble her daughters (albeit with longer hair).
The High Priestess is a cruel, unfeeling, ruthless and remorseless woman, who punishes those who make mistakes in a dangerous and violent manner, including her own daughters. She puts her children through intense and harsh training, even at a young age, and breeds them into becoming assassins and servants for the demon Aku.
She has no genuine concern for her children, and the closest thing to affection that she ever displays is to Ashi, whom she refers to as "My sweet little Ashi" during training. She only shows true love towards her husband Aku, who is worshiped as a god by both the High Priestess and her followers in the Cult of Aku. She lives to serve Aku and puts her daughters through assassin training as a result, explaining how the weak have no place with Aku.
In "Episode XCII", the High Priestess showed up during a ceremony conducted by the Cult of Aku, an all-female cult of devout worshipers of Aku. There, she gave birth to seven girls and put them through years of hard and intense training to make them into strong ninja assassins who will hopefully track down and kill Samurai Jack, Aku's long-time rival and enemy. After training them, she made them to kill the other cultists and gave them white female masks and weapons and sends them out into the world to kill the samurai.
In "Episode XCV", although the High Priestess didn't appeared physically, Ashi has a dream about her while unconscious, appearing as a nightmarish face demanding her to kill Jack. Later, before she can finish off an exhausted Jack, she sees a ladybug pass and remembers the time her mother squished one for "distracting" her from a sparring session, when Jack holds the ladybug he admires it and let it fly away.
In "Episode XCVI", when Ashi joins Samurai Jack, the High Priestess appeared to Ashi during the night as a vision in the moon telling her to kill Jack while he is sleeping, but Ashi denies to do it explaining to her mother that Jack saved her life and wants to know the truth. The High Priestess angered for Ashi's response then tells her that she has failed her.
In "Episode XCVII", during her quest for Jack who previously went missing with the Omen, Ashi approached a waterfall in the forest and there she remembered the time her mother indoctrinated her and her sisters by having them dive into burning ashes, "becoming one with the darkness" and giving them their signature "outfits". The High Priestess then pushed Ashi first into the pit, showing little care of the screams of pain from her daughter. Diving into the river, Ashi spend the rest of the night scrubbing off her ashes, letting her hair down and creating a new outfit from nearby foliage, finally putting the past behind her.
In "Episode XCVIII", the High Priestess decides to confront the samurai herself along with an army of soldiers while he's distracted with meditating on a mountaintop to retrieve his sword. Ashi manages to defeat all of the soldiers, but the High Priestess sneaks to the top and fires arrows at the samurai, however, Ashi stops them in time. Enraged, the High Priestess battles her final child, accusing her of betraying her deceased sisters by letting the samurai live. However, Ashi denies this and blames the High Priestess for their deaths, by feeding them lies and making their only purpose in life to kill Samurai Jack. When Ashi is dazed by crumbling debris, the High Priestess makes one final attempt to kill the samurai, though when Ashi throws one of her arrows at the High Priestess at the last second, it impales her through the spine and causes her to fall off the mountaintop. It's completely unknown if the High Priestess survived her impalation, but even if she survived that ordeal, she most likely was killed by the fall.
In "Episode C", The High Priestess is shown in a flashback in which she and her cult are visited by Aku. Aku notices the statue they constructed in his likeness and was impressed by the display. As a reward, he gifted them with a small fragment of himself within a chalice, to which the High Priestess drank shortly afterwards. Afterwards, the small bit of Aku caused her to become pregnant with Aku's children, thus giving birth to the Daughters of Aku.
In the series finale "Episode CI", after Jack successfully destroyed Aku in the past and Ashi was erased from existence for helping him to succeed in his mission, the High Priestess eventually never became the leader of the also wiped from existence Cult of Aku, as Aku was never there to have them to appreciate him nor to impregnate her with his essence. However, whether the High Priestess and her daughters (with a human father, of course) still exist in the new timeline or not is unclear, although it could be possible that she instead became a loving mother who loved her children as Aku never had any influence on her life.
The High Priestess drinking Aku's essence.
The priestess giving birth to seven daughters.
The High Priestess putting her clothes on with the help of 2 cult members.
"KILL THE SAMURAI!"
"Ashi. These frivolous distractions are poisons for your mission. They are not part of Aku's order."
The High Priestess in "Episode XCVIII".
The High Priestess making a last effort to kill Jack.
The High Priestess is one of the most depraved villains in the Samurai Jack series due to her remorseless and inhumane treatment of her children as well as having them beaten for things such as looking outside and showing compassion for each other. She is also easily one of the darkest characters in the show. She shares this distinction with Aku himself (though he did not actually choose to be evil even though he is the embodiment of all that is evil, unlike her) and the Dominator.
The High Priestess' fate after Jack destroyed Aku in the past is unknown, given the fact it was Aku who influenced her to become the terrible and abusive leader she was in his timeline, and that she died during her fight with Ashi, it's presumed that she never became evil in the current timeline created by Jack without Aku's influence or that she actually didn't exist like some of the other antagonists from Aku's timeline.
She shares the same alias as another female religious leader from Lightning Returns: Final Fantasy XIII and Babylon A.D..