Rivers are habitats favored by insectivorous bats. The Japanese large-footed bat (Myotis macrodactylus) is one of such species strongly associated with rivers. To clarify the population structure and migration pattern of M. macrodactylus, mitochondrial DNA (mtDNA) phylogeography was investigated in 15 subpopulations along three rivers (Tokachi, Ishikari and Teshio Rivers) on Hokkaido Island, northern Japan. Of 267 bats examined, nine mtDNA cytochrome b haplotypes were identified and separated into two major genetic clades, which did not reflect geographic distributions within and among the river regions. Principal component analysis of mtDNA haplotypes showed that most subpopulations in Ishikari and Teshio of the three river regions were grouped into one genetically related group, whereas those in the Tokachi river region were remotely related to those of the other two river regions. The results could be ascribed to no geographic barriers preventing bats' migration between the former two river regions. By contrast, it is possible that the Daisetsuzan and Hidaka mountain ranges play a role as effective geographic barriers against migrations of this species between the Tokachi and Ishikari/Teshio river regions.