The genetic population structure of the Japanese amblycipitid catfish Liobagrus reinii was investigated using partial sequences of the mitochondrial DNA cytochrome b gene, focusing on the pattern associated with the Central Highlands (the Fossa Magna region), the major geographic barrier for Japanese freshwater fish. Phylogenetic analyses revealed two highly divergent lineages within this species (clades 1 and 2), with each lineage further divided into three or four allopatric subclades (1-1, 2, 3, 4 and 2-1, 2, 3), suggesting that geographic isolation, such as mountain uplifting, was the major factor influencing population structure. One exception was the co-occurrence of two subclades (1-1 and 2-1) in several rivers of the Chugoku and Shikoku regions of western Japan, which would have resulted from a secondary contact between historically isolated populations via river capture. Subclade 1-2 haplotypes were widely distributed in the central area of Honshu Island (Pacific side) across the Central Highlands, where population and species differentiations are often evident in freshwater fishes. This pattern, along with circumstantial evidence, supports the theory that eastern populations from the Pacific side of Honshu originated from artificial introduction. Together with the western limit of the northeastern populations on the Sea of Japan side of Honshu, the role of the Fossa Magna region as a geographic barrier was emphasized also in this species. Based on the deep intraspecific divergence of the species and large differentiation from continental congeners, L. reinii was concluded to be an old member of the Japanese freshwater fish fauna, which extended its distribution over the Japanese Archipelago at an early time.