"Kono Naka ni Hitori, Imōto ga Iru!" centers on Shōgo Mikadono, a high school student who inherits his late father's massive corporation. As the new head of the Mikadono Group, Shōgo must navigate his newfound responsibilities while attending a prestigious academy filled with elite students. However, his father's will stipulates a peculiar condition: Shōgo can only assume full control of the company if he marries by his 18th birthday. Complicating matters further, Shōgo discovers that one of the girls at his school is his long-lost half-sister, though her identity remains a mystery.

The narrative unfolds as Shōgo interacts with several female classmates, each of whom could potentially be his sister. These include Konoe Tsuruma, a childhood friend and skilled martial artist; Miyabi Kannagi, a talented violinist with a reserved demeanor; Rinka Kunitachi, a cheerful and athletic girl; Mana Tendō, a sharp-tongued class representative; and Mei Sagara, a shy and introverted girl with a passion for art. As Shōgo grows closer to each of them, he must balance his personal feelings with the looming possibility that one of them shares his bloodline.

The story blends elements of romantic comedy, drama, and mystery, as Shōgo navigates his relationships while uncovering clues about his sister's identity. The series explores themes of family, identity, and the complexities of human connections, all set against the backdrop of high school life and corporate intrigue. The interplay between the characters' personal struggles and the overarching mystery drives the plot forward, creating a dynamic and engaging narrative.