The phylogenetic relationships between gobies of the genus Gymnogobius were analyzed using mitochondrial cytochrome b gene sequences, focusing on the species currently classified as G. taranetzi and G. castaneus that occur in Japan, South Korea, and Russia. Gobies of the two species collected at 12 localities in Japan, South Korea, and Russia formed a monophyletic clade (called the "castaneus species complex" here) with G. breunigii as the sister clade. Within the species complex, six lineages were recognized: (L1) G. castaneus from the Akigawa River, Tokyo, Japan; (L2) G. castaneus from Yuza, Yamagata, Japan; (L3) G. taranetzi from Russia and South Korea; (L4) G. castaneus from the Tonegawa River, Chiba, Japan; (L5a) G. taranetzi from Shimane, Japan; and (L5b) G. castaneus + G. taranetzi from the Japan Sea coast of northern Japan. The two local lineages of G. castaneus (L1 and L2) are highly divergent from the others. The Japanese populations of G. taranetzi have diverged from the continental G. taranetzi populations, while one mitochondrial lineage (L5b) is shared with G. castaneus of northeast Japan. Therefore, the current species G. taranetzi and G. castaneus as defined morphologically are polyphyletic, necessitating a taxonomic revision. The genetic differentiation of isolated local lineages and the evolution of taranetzi-and castaneus-type gobies have likely occurred repeatedly in brackish/freshwater habitats around the Sea of Japan. We discussed the time of divergence for these gobies based on a tree with the molecular clock assumption.