The game's plot will revolve around the personal stories and tragedies in the series. Its main focus is set on retelling the events of Resident Evil 2 and Resident Evil Code: Veronica, though a new chapter has also been confirmed. For the Resident Evil 2 segment, the player will take control of protagonists Leon S. Kennedy and Claire Redfield, with Ada Wong and Sherry Birkin acting as supporting characters. The Code: Veronica chapter will again feature Claire, accompanied by fellow Rockfort Island prisoner Steve Burnside. Albert Wesker is set to make an appearance in a flashback.
In addition to the retellings, there will be new aspects to the series' storyline that will be explored, such as a chapter starring Leon in previously untold events in South America. As the game's development began before the production of Degeneration and because of the different timeframe The Darkside Chronicles is set in, Kawata ruled out connections to the CG film and Resident Evil 5, though he hinted at the appearance of new characters and the exploration of the Birkins' and the Ashfords' family ties.