In 1202, an army led by the three vampire elders (Markus, Viktor, and Amelia) arrives at a Lycan-ravaged village. Viktor and Amelia capture their target, Markus' brother, William Corvinus, the first and most powerful werewolf. Despite Markus' defiance, Viktor orders that William be imprisoned in a secret location forever.
In the present day, vampire Selene takes Michael to a safe house so that she can confront the vampire regent Kraven; she knows that Kraven intends to kill Markus and plans to stop him. However, Markus has awakened before Kraven arrives. Markus kills Kraven and his men. Lorenz Macaro, an elderly man, sends in a team of "cleaners" to investigate the aftermath from the battle in the Lycans' lair. When Lorenz Macaro examines Viktor's corpse he finds a metal disc inside it which is the match to a pendant originally worn by Sonja. The other half of the pendant is now possessed by Selene and Michael after the deaths of Viktor and Lucian.
Markus tracks Selene and Michael down and attacks them, but they evade him and hide in a warehouse. There, Selene and Michael share their feelings and engage in sexual intercourse. Now knowing that the pendant is important to Markus, Michael and Selene set out to discover why Markus wants it. Selene recalls that she saw it as a child, but does not know its significance. They travel to the hideout of the exiled vampire historian Andreas Tanis who reveals that Markus was the first vampire; one of the three sons of Alexander Corvinus, the first immortal. Markus was bitten by a bat and became a vampire, while his twin brother William was bitten by a wolf and became a werewolf. The third son remained human and gave rise to a line of mortal descendants including Michael who became the first Lycan–Vampire hybrid. The first werewolves created by William were entirely animal and unable to assume human form. Due to William's destructiveness, Markus approached Viktor, who was a dying mortal warlord at the time, and offered to turn him and his army into immortal vampires in exchange for tracking down and stopping William, and in destroying those he had infected.
Viktor did not kill the brothers because he was deceived by Markus into believing that doing so would result in the immediate extinction of all other vampires and his Lycan slaves. Tanis reveals that Selene's father was the architect who built William's prison and that the pendant is a key to the latter. Viktor killed Selene's family as they knew the prison's location, but turned Selene into a vampire so the memory of the prison's location would be kept in her blood. Tanis then refers Selene and Michael to Macaro for help. After Selene and Michael leave Tanis' residence, Markus arrives and drinks Tanis' blood to learn Selene and Michael's location, killing Tanis.
Selene and Michael visit Macaro and discover that he actually is Alexander Corvinus. Alexander reveals that he has devoted his entire immortal life to keeping the Vampire-Lycan war a secret. However, he refuses to assist Selene in killing his sons. Then Markus arrives, impaling Michael and learns the location of William's prison by drinking Selene's blood. He mortally wounds his father and obtains the other half of the pendant, after deriding his father's refusal to help William and revealing that he intends with William to rule the world as the god-like master of a race of vampire-Lycan hybrids. On Alexander's bidding, Selene drinks his blood, enhancing her physical strength and healing abilities to a level equivalent to that of a hybrid. Afterwards, Alexander blows up his ship, killing himself.
Selene, aboard Corvinus' helicopter, leads his cleaners to the prison to confront and destroy Markus, but he has already freed William. A battle ensues in which William bites the cleaners who, as a result, are turned into werewolves. Michael, who is presumed to be dead and is carried aboard the helicopter, regenerates and joins the fight in his hybrid form, killing William by ripping his head off. Selene engages Markus in hand-to-hand combat, killing him by kicking him into the rotor blades of the cleaners' crashed helicopter. After the battle, Selene realizes that Alexander's blood has made her immune to the lethal effects of sunlight on vampires.