To clarify the genetic characteristics of the white-spotted char, Salvelinus leucomaenis, which inhabits the Shimoyosaku tributary (which is isolated from the main stream during the normal stage of water), we performed mitochondrial DNA cytochrome b region analysis and microsatellite analysis of 35 individuals sampled from both groups of the isolated tributary (Shimoyosaku) and the main stream. mtDNA cytochrome b region analysis confirmed both groups to have only one haplotype that has been observed in populations of the North Kanto area.Genetic diversity of the isolated group was lower than that of the mainstream group, and gene divergence was greater between groups, suggesting that gene flow between groups was extremely low.