This red-tongued pit viper (Gloydius ussuriensis) inhabits the Korean Peninsula, including inland areas as well as Baengnyeongdo, Jeju-do, and Gapado Islands. However, the taxonomic status of Gloydius ussuriensis populations, including their subspecies classification, remains unclear, as few phylogenetic or taxonomic studies have compared inland and island populations. To address this gap, we analyzed 513 individuals sampled between 2018 and 2022. Mitochondrial DNA revealed two major lineages: (1) inland, Baengnyeongdo, and Chinese populations and (2) Jeju-do and Gapado populations. Nuclear DNA analysis (K=3) identified three genetic groups, namely, inland, Baengnyeongdo, and Gapado, whereas the Jeju-do population exhibited an admixture of the inland and Gapado groups. Discriminant analysis of the 11 morphological traits indicated clear differences among the Baengnyeongdo, Gapado, Jeju-do, and inland populations. The snout-vent length was the longest in Baengnyeongdo and the shortest in the Gapado group. The ecological niche overlap between the populations was low (PI=0.4075). Based on these findings, three subspecies have been identified: G. ussuriensis ussuriensis, G. ussuriensis baengnyeongensis, and G. ussuriensis jejuensis. The Tsushima Island pit viper (G. tsushimaensis) from Japan, although classified as a separate species, showed genetic and morphological similarities to the Jeju-do-Gapado group. Further phylogenetic and taxonomic studies are needed for G. ussuriensis island populations in southwestern Korea, which were not included in this study.