"Genshiken Nidaime" is the sequel to the original "Genshiken" series, continuing the story of the Genshiken club, a college group dedicated to otaku culture, including anime, manga, and gaming. The narrative shifts focus to a new generation of club members while retaining connections to the original cast. The story begins with Madarame, a former member and now a salaryman, revisiting the club and finding it transformed under new leadership. The club is now predominantly female, with Hato, a male cross-dresser, taking a central role alongside other new members like Yoshitake and Yajima.

The series explores the dynamics of the club as it navigates the challenges of maintaining its identity while adapting to the changing interests and personalities of its members. Hato's cross-dressing and his complex relationship with his identity become a significant plot point, as does his growing bond with Madarame, who struggles with his own feelings of isolation and unrequited love. The interactions between the new and old members highlight the evolving nature of otaku culture and the personal struggles of those who embrace it.

As the story progresses, the club prepares for Comiket, a major event in the otaku calendar, which serves as a backdrop for character development and interpersonal conflicts. The narrative delves into themes of self-acceptance, friendship, and the pursuit of passion, with a focus on how the characters reconcile their personal lives with their otaku interests. The series balances humor and drama, providing a nuanced portrayal of its characters and their relationships within the context of a niche subculture.