Pterogobius elapoides and Pterogobius zonoleucus are common free-swimming gobies found in rocky and weedy shores along the temperate coast of Japan. We collected individuals of both species from 23 locations around the coast of Japan and compared the mitochondrial nucleotide sequences of two gene regions, CytB and ND2. Phylogenetic trees constructed using the neighbor-joining, maximum parsimony, and maximum likelihood methods consistently indicated that all 125 samples of the two species, which are collected from a variety of locations in Japan, can be clearly divided into the following four clades: "Pacific P. elapoides" (Pa-ela), "Sea of Japan P. elapoides" (SJ-ela), "Pacific P. zonoleucus" (Pa-zon), and "Sea of Japan P. zonoleucus" (SJ-zon). These four monophyletic clades were supported with very high bootstrap values. Although Pa-ela and SJ-ela composed a monophyletic clade, it is noteworthy that the two clades of P. elapoides also formed a monophyletic group together with SJ-zon with a bootstrap value of 95% and 97% by the maximum likelihood and neighbor-joining methods, respectively. We observed several morphological differences between Pa-ela and SJ-ela, including; 1) six dark bands on the body in the former versus seven dark bands in the latter and 2) more pectoral-fin rays numbering 21-24 (mode 22) in the latter compared to the former (19-22, mode 21). Furthermore, the scatter plots of scores on principal components 1 and 2 based on the morphometric characters roughly separated the populations from each other. Moreover, we documented the following morphological differences between Pa-zon and SJ-zon for the first time; 1) six light bands on the body in the former versus five light bands in the latter and 2) the light bands from both eyes forming a complete U-shaped marking on the occipital region occurred in 55% of the specimens in the former versus 16% in the latter. However, no significant differences were found in the morphometric characters between the two populations of P. zonoleucus. The estimated divergence time of the two P. zonoleucus populations was 15.06+/-2.72 (mean+/-1 S.E.) times earlier than that of the two P. elapoides populations. However, the morphological differences between the two populations of the former were much smaller than those of the latter. An explanation for this obvious discrepancy between morphological and molecular features is proposed from an evolutionary point of view.