Abstract: Labeoninae belongs to Cyprinidae of Cypriniformes , approximate 26 genus and 300 species. To study the phylogenetic relationships of Labeoninae fishes in Cyprinidae, the Phylogenetic Tree was reconstructed based on sequences of mitochondrial 16S rRNA. The 16S rRNA partial sequences of three species of Labeoninae were amplified by Polymerase Chain Reaction method, which were comprised of 361~362 bp respectively. 364 sites were found by relative seuqence aligment of which 94 sites were information site. Using neighbor-joining of Mega 2.1 software reconstructed the molecular Phylogenetic Tree, the results indicated Labeoninae fishes might not be monophyletic population, C.molitorella and E.bicolor of Labeoninae assembled together with Cyprinus carpio and Carassius auratus firstly, and formed sister group, then assembled together with C.mrigala and L.rohita, which suggested that C.molitorella was genetically more relative to Cyprinidae than Labeoninae.