Abstract: The Odontobutis are valuable small freshwater fishes restricted to the East Asia, but their specific validities and phylogeny has been controversial for a long time. To determine interspecific relationships and evolution of the genus Odontobutis, partial sequences of mitochondrial 12S rRNA genes of all putative species of Chinese Odontobutis fishes (O. sinensis, O. haifengensis, O. yaluensis, and O. potamophila) were sequenced and subjected to phylogenetic analysis with the inclusion of two Japanese species (O. obscura and O. hikimius) and available taxa from Eleotridae obtained from GenBank. The resultant mitochondrial DNA sequence were 690bp long with 34 indels, of which 258 sites were variable and 201 sites were parsimonyinformative, and average transition/transversion ratio was 3.0, suggesting that 12S rRNA gene was a suitable molecular marker to be used in studying the interspecies relationships within Odontobutis species. The genus Odontobutis formed a monophyletic group with 100% bootstrap support, and p-distance model-based pairwise genetic distances within each putative species ranged from 0.000-0.024, and those among the six Odontobutis species were between 0.058 to 0.133, which agree with Wu＇ s taxo-nomic treatment of the genus, i.e., that O. obscura and O. sinensis were different species, and there were 4 species of Odontobutis in China. In phylogenetic tree, while O. haifengensis and O. potamophila were closely related, and O. hikimius is sister group, and the phylogenetic relationships among the remaining taxa of the genus were not well resolved. As Chinese Ondontobutis had not been sampled extensively and intensively in the present study, whether there are new Odontobutis species or not yet to be further studied. Chinese species and Japanese taxa of the genus Odontobutis were not resolved as monophylogentic group respectively, it is deduced that the common ancestors of the genus Odontobutis might be widely distributed in China, Korea peninsula and Japan, diverged from 4.9 to 6.5 million years B.P. in middle Pliocene, and their phylogenetic process might be explained by vicariant hypothesis.