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Six months after the events of Underworld: Evolution, Selene (Kate Beckinsale) was captured by humans during the purge, when she and Michael were planning on leaving. Eventually both government officials and the public learn about the existence of vampires and Lycans. A crusade and all-out war has begun: study and eradicate. After being imprisoned in cryogenic suspension for 12 years, Selene manages to escape the facility. During the same time, another subject has also escaped the same facility that Selene was in. That subject was Eve (India Eisley), her hybrid daughter who is the key to ending the war by "curing" vampires and Lycans of their "infection". After escaping she sees flashes of someone's point of view and, believing it is Michael, she follows the flashes, leading her down into a garbage chute. She confronts a young vampire named David (Theo James) who has been following her, when they realize that Lycans live there underground. Seeing flashes of Lycans hunting the person she is connected to, she eliminates many of them until they flee and she confronts the being who is not Michael, but Eve after getting her out of the tunnels. They are chased in a van by the three Lycans and after a lengthy chase Eve rips one Lycan's head in half while David and Selene shoot and stab the other two. On the way Selene realizes that Eve is not healing from a wound she suffered during her fight and is growing weak. David takes them to his coven.
At the coven most of the vampires are less than thrilled that David brought the infamous Selene the lover of a Lycan to their coven, especially his father. However, they agree to help the weakened child which they soon realize is due to her not feeding in a long time. After drinking some blood her wound heals and Selene, after seeing Eve's eyes after the feeding, sees that she has Michael's hybrid eyes and realizes she is her's and Michael's daughter. Seeing that the coven does not want her there, she gets ready to leave. David, not wanting her to leave, tries to convince her to stay and help them start an offensive against the humans by training some into Death Dealers. Before she makes up her mind the coven comes under attack by Lycans who were thought to be almost extinct. The vampires are slaughtered one after another until Selene joins the fight and kills many of the Lycans. She has David take Eve to safety while she deals with the rest of the Lycans. Going to the main hall of the coven, she comes upon a massacre of mostly vampires with few Lycan corpses and fights an 18 foot tall super Lycan which is more than 4 times the size of any other Lycan. Fighting it with a silver bladed weapon she breaks a piece off on its back, and is shocked to realize that it heals instantly and that the silver had no effect. She is knocked out and awakens later to find that David's father gave Eve to the Lycans to placate them into leaving. David dies after confirming this and his father blames Selene for all that happened, before Selene cuts a hole into David's heart and uses her blood to restart his heart. She then confronts a human detective named Sebastian to confirm her suspicions of the corporation dedicated to killing off the immortal species.
After seeing the evidence Selene discovers that the corporation is actually run by the Lycans - who have been posing as humans in order to trick the world's governments into believing the Lycans are extinct - and that they intend to use Eve to perfect their race and develop immunity to silver, thus allowing the Lycans to conquer both vampires and humans. Seeing the dangers Selene and the Detective team up to stop them and save Eve. Killing the Lycan guards with silver gas grenades and silver bullets, she comes across Subject 0, realizing it is Michael and, being short on time, shoots the glass to weaken the containment unit in order to free Michael before going to stop the Lycans from leaving with Eve. Using her incredible strength she flips the van and then comes face to face with the Super Lycan again, whilst Eve fights the Lycan doctor that held her. Eventually Selene goes down a hallway where the Super Lycan cannot fit, forcing it to morph back into a human, allowing Selene to impale him with her arm. Withdrawing her arm, the Super Lycan still heals instantly but Selene is counting on this and throws a grenade pin at him. Realizing too late that she has put a grenade inside him, the super Lycan claws in vain at his stomach before it explodes, killing him, while Eve rips out the throat of the Lycan doctor. Going back inside they find that Michael's cryo tank is empty; Eve then gets a flash of the roof where they go, but Michael is no where in sight. Selene states that the world will be hunting Michael just as they hunted Eve.