Abstract: In the present study,thirty-six individuals of Acheilognathus macropterus were collected from the Heilongjiang River,the Changjiang River,and theNandujiang River. Partial mitochondrial cytochrome b gene region(636 base pair)was sequenced to these samples and 22 haplotypes were found.With A 1chankaensis and A1tokinensis as outgroups,their relationships were analyzed.The p-distances were calculated with Mega software,and molecular phylogene-tic tree was constructed using the neighborjoining(NJ)method.Proportions of main morphological characters were compared as well.P-distances showed that the genetic differences in A1macropterus samplesweremuch smaller than those between these samples and the outgroups.Molecular phylogenetic tree showed that samples with barbels and those without barbels were intermingled. There was no distinct difference in proportions of morphological characters.The results suggested that samples with barbels and those without barbels(formally identified as A ltaenianalis)are the same species,A ltaenianalis is a junior synonyms of A1macropterus. The thirty-six individuals were grouped into 5clades,and the positions of the samples in the clades were corresponded with their geographical distributions.Among the 5 clades,clade 1and clade 5 included specimens from Heilongjiang River and Nandujiang River respectively.The specimens from Changjiang River scattered into clades 2,3,and 4,and there were distinct genetic differences (>5%)among them.Interestingly,the distributions of the 21 samples in these three clades were not correlated to their geographical distributions.It is postulated that these genetic differences were due to bitterling.s mate choice,the prozygotic isolation. The genetic differences between the fish from Nandujiang River and those from the mainland indicated they were separated early.However, the small genetic differences among the samples and their positions of the fish from Heilongjiang River in themolecular phylogenetic tree indicate that fish from Heilongjiang River might disperse from Changjiang River very late.