In order to clarify the genetic diversity and differentiation of Odontobutis potamophila from different geographic populations in Anhui Province, 142 individuals from the Yangtze River and Xin'an River Basin were collected. The mitochondrial genes were chosen as the marker to analyze. A total of 1 291 bp marker was obtained which contained 52 variable sites and defined 15 haplotypes. The genetic diversity of four populations was higher than intra-populations, and the molecular variation was mainly from inter-populations. All populations gathered three branches that reflected the geographical distribution of the species, and no expansion had occurred. This study revealed a high inter-population differentiation level of Odontobutis potamophila and also suggested a high conservation and selective?breeding value between each population.