The Ogasawara Islands, isolated from the continent throughout their geological history, harbor abundant endemic species. However, 32% of the native plant species are on the Japanese Red List, signaling their endangered status. Among these endangered species, Calanthe hoshii stands out as highly susceptible to extinction. Two wild and 89 ex situ individuals were alive in 2020, but the last known wild individual died in October 2021, indicating this species may have become extinct in the wild. Despite the conservation efforts under the Act for the Conservation of Endangered Species of Wild Fauna and Flora, the lack of essential information regarding genetic diversity and population structure among the remaining individuals poses a significant obstacle to developing effective conservation measures. In this research, we conducted a genetic analysis of the wild and ex situ individuals, which revealed notably reduced genetic diversity between individuals, with individual heterozygosity levels (1.0 × 10−5) measuring one‐ninth of those observed within a closely related species Calanthe triplicata (1.5 × 10−4). Comprehensive genetic analysis revealed that C. hoshii consisted of three genetic clusters of different sizes: cluster 1 comprised 95% of the total population, while clusters 2 and 3, with few individuals, were only found in the ex situ populations at Koishikawa Botanical Garden. Since extant C. hoshii maintains a remarkably low genetic diversity at both individual and population levels, it is necessary to consider future management strategies, such as artificial breeding among different clades identified in this study, to safeguard the stability and resilience of this species.