Abstract: This study presents the mitochondrial 16S rRNA partial sequences representing 13 genera of Chinese freshwater mussel Unionidae. Alignments were conducted with the help of Clustal X.After alignment,there were 305-320 sites. Sequence variations were analyzed with Mega 2.0 software,one hundred and eight sites were variable,among which there were seventy seven phylogenetically informative sites."Pairwise distances" were computed among the 13 genera with Mytilus edulis designated as outgroup. Only one molecular phylogenetic tree was obtained using Neighbore Joining method of Mega 2.0.The bootstrap analyses (1000 replications) were performed to test the confidence of nodes. According to our results,Unionidae recorded from China group to three clades: a basal Hyriopsis + Solenaia + Lamprotula + Ptychorhynchus clade,an Acuticosta + Unio + Ar2conaia + Cuneopsis + Lanceolaria + Schistodesmus + Arconaia calde and an Anodonta + Cristaria clade. Therefore,Chinese unionids should be divided into three subfamilies: Ambleminae( Hyriopsis + Solenaia + Lamprotula + Ptychorhynchus ),Anodontinae( Anodonta + Cristaria ) and Unioninae ( Acuticosta + Unio + Arconaia + Cuneopsis + Lanceolaria + Schistodesmus + Arconaia ),which also demonstrates the existence of Ambleminae in China. Thereinto,the analysis of mitochondrial 16S rRNA indicates that Solenaia and Lepidodesma,which were placed Anodontinae before,should be moved to the subfamily Ambleminae and Unioninae respectively. To more accurately reflect the phylogeny, Hyriopsis, Lamprotula and Ptychorhynchus should be moved to the subfamily Ambleminae.