The genetic relationships in a subspecies of white-spotted char, Salvelinus leucomaenis japonicus Oshima, distributed in central Japan, were examined based on the DNA sequences in the mitochondrial cytochrome b region. A total of 12 haplotypes were recognized; haplotypes 2 and 5 were co-dominant. Two large clades were observed in a genetic tree. The Hidaka, Yahagi, and Ibi rivers constructed only the larger clade, whereas the Yogo and Sagami rivers constructed only the smaller clade. A small clade of haplotypes 3, 8, and 10 comprising the samples from the Hidaka and Yahagi rivers and almost all samples from Kiso River was separated deeply. In addition, haplotype 8 from the Nomugi (1446 m) site was estimated to be the newest lineage. However, there were no significant correlations between geographical and genetic distances. These results suggest that this subspecies has multiple origins, its genetic structure is determined by other factors than the river basin, and geological events were scarcely involved in its range expansion.