Cryptic species are common in gobioid fishes, as revealed by recent molecular studies. This study collected molecular and morphological data to delimit species boundaries and to reveal the historical biogeography of Odontamblyopus lacepedii sensu lato by sampling 87 specimens from 16 locations in the northwestern Pacific. Phylogenetic trees based on mitochondrial NADH dehydrogenase subunit 5 sequences identified three major clades. Clear morphological separations among these clades were detected by examining morphological characters used in taxonomic diagnosis. Therefore, these clades were delineated as three separate species: O. lacepedii sensu stricto (in the East China Sea, Yellow Sea, Gulf of Bohai, and Ariake Bay); O. sp. (in the southern East China Sea and southern Yellow Sea); and O. rebecca (in the Gulf of Tonkin, northern South China Sea, and southern East China Sea). Estimated divergence times were 0.61 +/- 0.15 Ma between O. lacepedii and O. sp., and 2.31+/-0.36 Ma between O. rebecca and O. lacepedii+O. sp. These findings indicate that isolation of marginal seas and habitat fragmentation during major falls in sea level in the late Pliocene and Pleistocene may have been responsible for genetic breaks among Odontamblyopus species in the northwestern Pacific.