‘Hariyo’ comprise the southernmost freshwater populations of the threespine stickleback, Gasterosteus aculeatus species complex, distributed in central Honshu, Japan. Two geographic groups (Gifu and Shiga) of the ‘hariyo’ have been recognized as differing from each other in some morphological and ecological features. In order to elucidate the genetic characteristics and phylogenetic position of these populations, partial sequences of the mitochondrial cytochrome b gene were compared in 123 specimens from 10 ‘hariyo’ populations, and eight other freshwater and anadromous populations of threespine stickleback in Japan. Phylogenetic analysis resolved 22 haplotypes in a single most-parsimonious tree. In contrast to previous allozyme analyses, the haplotype tree indicated that ‘hariyo’ populations are monophyletic. Each of two geographic groups of the ‘hariyo’ was also shown to be nearly monophyletic. The two geographic groups differed from each other in sequence by an average of 0.47% and from other populations by 0.70%. Based on a molecular clock, constructed from fossil records and mtDNA genetic distances of Gasterosteus and Pungitius, it was estimated that the two groups of ‘hariyo’ differentiated from the other threespine stickleback populations and also from each other in the middle Pleistocene. The results suggested that each of the two groups of ‘hariyo’ is a distinct evolutionarily significant unit with unique genetic features, as well as morphological and ecological characteristics.