Genetic relationships among a subspecies of white-spotted char, Salvelinus leucomaenis pluvius, distributed in the border area of three river basins in the eastern Chugoku region of Japan (the Sendai River, flowing into the Sea of Japan, and the Yoshii and Chigusa rivers, both flowing into the Seto Inland Sea), were investigated based on mitochondrial DNA cytochrome-b gene sequences. A total of 7 haplotypes were recognized and there were 8 polymorphic sites in a 447 bp fragment. Some haplotypes were shared by fish in two adjacent basins across passes, and some were confined to restricted basins. One haplotype estimated to be the oldest could be connected to ‘Gogi’, S. leucomaenis imbrius. Another haplotype, estimated to have derived in the most recent period, was observed in more than half the samples. There was a significant positive correlation between the geographic and genetic distances. These results suggest that genetic distance was more strongly determined by geographic distance than by river-basin location, and may support the possibility of range expansion of char by taking advantage of geologic events.