In this study, the partial mtDNA 16s rRNA gene and the S7 nuclear intron were used to resolve the phylogenetic relationship of four morphologically similar members of Ostorhinchus, including two colour morphs of Ostorhinchus properuptus (lined and dotted types),
Ostorhinchus cyanosoma and Ostorhinchus holotaenia. Two different tree‐building methods, maximum likelihood (ML) and a Bayesian‐based method, yielded trees with highly similar topologies and high bootstrap values and posterior probabilities on the main nodes. Two
main clades were recovered, suggesting that O. properuptus dotted type is closely related to O. holotaenia, and O. properuptus lined type members with O. cyanosoma. In addition, the genetic distance between the dotted and lined types of O. properuptus (0·299)
was 23‐fold higher than that between the dotted type and O. holotaenia (0·013). The dotted type can be identified by genetics as slightly differentiated in Japanese waters. It is treated as a population of the widespread species O. holotaenia.