Abstract: In order to clarify the differences between the Odontobutis sinensis and O. potamophilus, population genetics were studied based on Cyt b gene and morphological characteristics were investigated using traditional morphological character measurement and truss method. 21 haplotypes were identified for 128 individuals of O. sinensis from 3 locations, and 28 haplotypes for 195 individuals of O. potamophilus from 4 locations. Haplotype diversity and nucleotide diversity of O. sinensis were 0.868 and 0.00407, respectively, while those of O. potamophilus were 0.910 and 0.02000. Phylogenetic analyses for all haplotypes based on BI and ML methods revealed that all Odontobutis species form a monophyletic group. Results showed that O. sinensis has 2 lineages and O. potamophilus consists of 3 lineages, with an obvious pattern of geographic distribution. AMOVA results indicated that the genetic differentiation (F_ST) among geographical populations of O. sinensis ranged from 0.463—0.845, while for O. potamophilus, it ranged from 0.653—0.930, suggesting significant genetic differentiation among populations. This finding implies that the most probable geographic differentiation of the populations. Mismatch distribution and neutrality test revealed that XN population of O. sinensis have experienced the population expansion event, and it may happen at 0.20 Ma. Slight differences in the results of historical dynamic analyses across methods suggest a relatively complex demographic history of Odontobutis. Morphological analysis revealed distinct variations in O. sinensis and O. potamophilus. Principal components analysis showed that most individuals of two species were separated. Then the discrimination functions of O. sinensis and O. potamophilus were set up by stepwise discriminant analysis with an accuracy rate of 95.7% (P<0.01). The eight components with the highest contributions to principal components analysis and discriminant analysis were related to the characteristics of fin and trunk. This quantitative divergence (Q_ST) was not significantly correlated with the genetic differentiation (F_ST), and the degree of F_ST was significantly high than that of Q_ST, which suggested that phenotypes were favored and stable selection occurred in different populations.