Abstract: In order to scientifically carry out the artificial breeding and release protection work of Coreius guichenoti, 199 samples of six populations of C. guichenoti were collected from the upstream and middle reaches of the Yangtze River (Yichang, Three Gorges Reservoir area, Yibin, Yongshan, Qiaojia, and Panzhihua). A total nine polymorphic tetranucleotide microsatellites of the six populations were analyzed in this study. The results demonstrated that all the nine microsatellite loci were highly polymorphic, and the six populations had high genetic diversities.  AMOVA analysis revealed there were no significant differences in the six populations. Nevertheless, Structure analysis showed that the six populations could be divided into two groups. One group included some individuals from the Three Gorges Reservoir population and some from the Yibin population, while the other samples were involved in another group. The results difference between AMOVA and structure analysis might be due to that, all samples in each population were identified as a single factor in AMOVA, but structure analysis recognized the samples proportion in each population. Therefore, in order to avoid the occurrence of genetic background pollution events, it is necessary to do the genetic management of artificial breeding of C. guichenoti and analyze their genetic structure of the released stock and nature population