"Kono Naka ni Hitori, Imōto ga Iru!" centers around Shōgo Mikadono, a high school student and the heir to the wealthy Mikadono conglomerate. Following his father's passing, Shōgo is informed that he must marry one of his potential fiancées to inherit the family fortune. The condition, however, is complicated by the revelation that one of these fiancées is his long-lost younger sister, whose identity remains unknown. To uncover her identity, Shōgo transfers to a prestigious private academy where all the candidates attend, intending to interact with them and solve the mystery.

The story unfolds as Shōgo navigates his relationships with the five potential fiancées, each of whom has a distinct personality and background. These include Konoe Tsuruma, a childhood friend and skilled martial artist; Miyabi Kannagi, a reserved and intelligent class representative; Rinka Kunitachi, a cheerful and athletic girl; Mana Tendō, a wealthy and confident heiress; and Mei Sagara, a shy and introverted girl with a passion for art. As Shōgo spends time with each of them, he uncovers their individual struggles and aspirations, all while attempting to determine which one might be his sister.

The narrative blends elements of romantic comedy, drama, and mystery, with Shōgo's interactions often leading to humorous and heartfelt moments. The series explores themes of family, identity, and the complexities of relationships, as Shōgo grapples with his feelings for the candidates and the moral dilemma of potentially falling for his sister. The mystery of the sister's identity drives much of the plot, with clues and revelations gradually emerging as the story progresses. The dynamic between the characters and their evolving relationships form the core of the series, creating a balance between lighthearted romance and the underlying tension of the central mystery.