The Tsushima salamander, Hynobius tsuensis, is a lotic-breeding species endemic to Tsushima Island, Japan. We investigated genetic variation in this species using samples covering all known localities on the island. Phylogenetic analyses based on mitochondrial cytochrome b gene sequences divided H. tsuensis populations into two genetic groups: true H. tsuensis and an unknown species (Hynobius sp.) showing a closer relationship to Hynobius nebulosus than to H. tsuensis. The genetic distance between the two groups was greater than the genetic distances between different species of Hynobius. Both groups were found to breed in mountain streams, sometimes in the same streams syntopically. Population structure analysis based on several nuclear microsatellite loci strongly indicated reproductive isolation between the two groups in the syntopic localities. This is the first report confirming the syntopic occurrence of two lotic salamanders of Hynobius on a small island. We propose that the lotic breeding habits of the two salamanders were independently acquired.