Darwi is the Mother Superior of the Bene Gesserit, tasked with leading her order while in hiding from the violent Honored Matres. She oversees the terraforming of the planet Chapterhouse, managing a difficult balance between the emotional repression typical of her order and her own deeply hidden compassion, which she personifies as her inner "Sea Child." She is a descendant of the Atreides bloodline and the daughter of the original military commander she recently ordered cloned. Her leadership style heavily favors long-term strategy over rigid tradition.

Duncan is the latest in a millennia-spanning line of gholas originally cloned from a legendary swordmaster. Held prisoner by the Bene Gesserit, he possesses memories of all his previous ghola iterations and operates secretly as a highly capable Mentat. His deep cynicism toward the Sisterhood is complicated by his intense, mutual bond with an Honored Matres captive, which prevents him from simply escaping.

Murbella is a former Honored Matres captive held by the Bene Gesserit. She possesses exceptional physical abilities and advanced sexual imprinting techniques, which originally resulted in her capture when a bonding attempt backfired. She undergoes rigorous training to become a Reverend Mother, forcing her to reconcile the aggressive instincts of her past with the disciplined control of her captors.

Miles is an artificially created clone of the late, legendary Bene Gesserit military commander. Raised under the watchful eye of the Sisterhood, he possesses the genetic potential of his predecessor but lacks his memories. He exhibits prodigious physical and mental skills early in life, making him both a valuable asset and a potential risk to those managing his education.

Sheeana is a woman of Fremen descent possessing the rare ability to control sandworms. She serves as an important figure in the Sisterhood's plans for ecological manipulation and religious influence. Despite her value to the Bene Gesserit, she harbors her own ambitions and maintains covert alliances that challenge the order's strict authority.

Dama is the leader of the aggressive Honored Matres, often referred to by the Sisterhood as the "Spider Queen." She commands a massive, violent force that has conquered much of the Old Empire. She aggressively seeks to capture and torture Bene Gesserit members to extract the secrets of their biochemical control and political longevity.

Scytale is the last surviving Master of the Bene Tleilax, an order of genetic engineers wiped out by the Honored Matres. He barters his knowledge of axolotl tanks and cloning in exchange for the Sisterhood's protection. He secretly carries a nullentropy capsule containing the genetic material of countless historical figures, viewing it as his ultimate bargaining chip.

Lucilla is a Bene Gesserit Reverend Mother carrying the shared memories of millions of Sisters from the destroyed planet Lampadas. Hunted relentlessly by the Honored Matres, she prioritizes passing on her vast reservoir of knowledge to ensure the cultural continuity of her order, even at the cost of her own safety.

The Rabbi is a leader of a secret community of Jews living in hiding on the planet Gammu. He maintains a longstanding mutual assistance pact with the Bene Gesserit. He must carefully balance the survival of his people against the demands of the Honored Matres and the desperate needs of the Sisterhood.

Rebecca is a "Wild" Reverend Mother who belongs to the secret Jewish community on Gammu. She has survived the equivalent of the Spice Agony without formal Sisterhood training, giving her access to ancestral memories. She takes on the massive burden of preserving the lost minds of the Lampadas Sisters.

Logno is a grand dame within the Honored Matres hierarchy and a close advisor to Dama. She oversees the management of superweapons and interrogations while quietly assessing the weaknesses of her superior. She acts as a conduit for military intelligence regarding the Bene Gesserit and other threats.

Tamalane is a senior Reverend Mother serving on Odrade's inner council. She provides logistical support and harsh tactical assessments regarding the impending war. She often accompanies Odrade on vital excursions across Chapterhouse, offering a grounding, traditional perspective on the rapid changes facing their home.

Bellonda is a Mentat and senior Reverend Mother on the Bene Gesserit council. She manages the extensive archives and handles the grim mathematics of the order's survival strategy. She is deeply suspicious of the male prisoners held on the no-ship and frequently advocates for their execution to eliminate potential risks.

Dortujla is a Reverend Mother who brings crucial information regarding a potential alliance with mysterious forces from the Scattering. She volunteers for highly dangerous diplomatic missions, acting as bait to draw the Honored Matres into negotiations.

Streggi is an acolyte and personal assistant to Odrade. She accompanies the Mother Superior on inspections of the changing Chapterhouse environment and receives ongoing instruction in history, morality, and leadership from her commander.

Daniel is an elderly face dancer who observes the events unfolding in the Old Empire from afar. He represents the mysterious forces from the Scattering, maintaining a strange, detached interest in the genetic capabilities and movements of the fleeing factions.

Marty is an older face dancer working in tandem with Daniel. She monitors the escape trajectories of various ships and expresses a calm, detached interest in the genetic subjects moving through uncharted space.