It is increasingly accepted that conservation work should consider the evolutionary history of target species. Fishes in the subfamily Acheilognathinae, family Cyprinidae, are, with the exception of three species exclusively distributed in Europe, restricted to Asia and show a distinct spawning behavior in laying their eggs in gill chambers of freshwater mussels. At present, many of the 70 species recognized in this group are facing with serious population decline in China and Japan, and their phylogenetic relationships are not well resolved. In the present study, based on mtDNA cyt b and 12S rRNA gene sequences, we reconstructed a more detailed species-level phylogenetic tree of this group, and assessed species conservation priorities based on their evolutionary distinctiveness. Molecular phylogenetic analyses showed that the Acheilognathinae contains two major clades: Acheilognathus clade and Tanakia-Rhodeus clade. Based on this phylogenetic result, conservation priority analyses were conducted using ED (evolutionary distinctiveness)/HED (heightened evolutionary distinctiveness), and EDGE (evolutionary distinctiveness and global endangeredness)/HEDGE (heightened evolutionary distinctiveness and global endangeredness) methods. The results suggested that T. himantegus, T. lanceolata, A. gracilis, A. imberbis, T. tanago, and A. longipinnis should be ranked as the top-priority species for conservation. According to our results, we also discussed the current conservation efforts of the bitterling fishes and gave suggestions for future work.