Intra- and interspecific phylogenetic analysis of Dolly Varden Salvelinus malma and white-spotted charr Salvelinus leucomaenis throughout Hokkaido Island was conducted using nucleotide sequences of the mitochondrial DNA control region and three nuclear DNA markers [ribosomal DNA (ITS-1), microsatellite (u-85), SINE (Fok-223)]. A total of 19 mtDNA haplotypes from 271 individuals of Dolly Varden, nine haplotypes from 134 white-spotted charr were defined, and identified two well-supported monophyletic clades for each species. Two haplotypes of Dolly Varden, however, were clustered together in the white-spotted charr clade. Those haplotypes were found only from several rivers in the Shiretoko Peninsula, and a neighbouring river of that region. Analyses of ribosomal DNA and Fok-223 loci revealed that both species are characterized by having completely species-specific diagnostic sequence and fragment patterns. Dolly Varden of the Shiretoko populations are typically allopatric in distribution where white-spotted charr do not currently occur. In addition, incongruence in genetic relationships between mtDNA and nuclear DNA markers give strong evidence of historical mtDNA introgression between Dolly Varden and white-spotted charr. Some white-spotted charr diagnostic alleles in a nuclear microsatellite locus (u-85) were found in some Shiretoko Dolly Varden populations, suggesting that introgressive hybridization might have also occurred in relatively recent contact and potentially ongoing evolutionary event. The present study presents an example of historical hybridization and introgression at the southernmost distribution limits of Dolly Varden.