The phylogeographic patterns of freshwater fishes provide clues for understanding the relationships between geological events and biota formation. In central Honshu, Japan, many mountain ranges (e.g., the Fossa Magna) divide watershed areas, resulting in long-term biogeographical barriers to the freshwater fishes. Almost all of the freshwater fishes from the upland areas, however, occur naturally in more than one river system. Such ichthyofaunal similarity among river systems is hypothesized as having resulted from headwater river captures. Accordingly, to clarify the processes behind present-day ichthyofaunal patterns in the upland area of central Honshu, the present study investigated the mitochondrial phylogeographic pattern of upstream fat minnow, Rhynchocypris oxycephala, distributed in mountainous streams in western Japan. The phylogenetic analyses, using mitochondrial cytochrome b gene sequences (1,140 bp) obtained from 451 individuals from 86 sites throughout the species distribution range, indicated eight major clades in Japan. Of these, the Biwa-Tokai clade was distributed in the major area of central Honshu, and divided into three geographically differentiated groups: (i) Lake Biwa group in the tributaries that flow into Lake Biwa; (ii) Ise Bay group in the Pacific drainages that flow into Ise Bay, and (iii) Hida group in the Japan Sea drainages. These groups were isolated by watershed areas in the IbukiSuzuka and Hida Mountains. However, haplotype distributions in the headwaters of the Shou (Japan Sea side) and Nagara Rivers (Pacific side) suggested former dispersal of freshwater fishes from the Pacific to Japan Sea drainages via river capture in the Hirugano Highlands watershed area. Similar distributions of mtDNA haplotypes observed in three other headwater areas also suggested previous river captures. During the present study, however, some mtDNA haplotypes collected from Gifu, Shizuoka and Kanagawa Prefectures were found to be identical to those in the Lake Biwa group, suggesting that artificial transplantations of R. oxycephala had occurred in several areas.