We analyzed partial sequences of cytochrome b (cyt-b), a mitochondrial DNA (mtDNA) gene, to determine the genetic relationships between six horsehead fish species: Branchiostegus japonicus, Branchiostegus albus, Branchiostegus auratus, Branchiostegus argentatus, Branchiostegus wardi, and an unidentified Branchiostegus species. The specimens were collected in Korea, China, Japan, and Vietnam. We compared their molecular phylogenetic relationships inferred from mtDNA cyt-b sequences with an osteological analysis. The unidentified species, B. sp., was similar to B. albus in terms of the lack of triangular silver-white dot at the posterior region of eyes (vs. large one present in B. japonicus), but was also similar to B. japonicus in terms of the presence of a straight-shaped first hemal spine (vs. a curve-shaped hemal spine in B. albus). Analysis of the mtDNA cyt-b sequences indicated that the smallest estimated sequence divergence was between the B. japonicus and B. sp. (0.70-0.94%), whereas the largest difference was between B. auratus and B. argentatus (23.06-23.36%). Both the maximum parsimony and maximum likelihood trees showed that the B. sp. was closely clustered with B. japonicus, and that B. auratus was most distant from the other species. When comparing the osteological characters, UPGMA tree showed that the B. japonicus and B. sp. were the most closely clustered species, and B. auratus was the most distantly clustered fish relative to the other species. The shape of the nasal, otolith and first hemal spine was informative for distinguishing B. auratus from the other species. These osteological differences were consistent with the differences in mtDNA.