As a small economic fish for human consumption, as well as a bait for large carnivorous fish and Neophocaena phocaenoides, Coilia brachygnathus occupies an important position in the food chain. Under the influence of various factors such as overfishing, environmental pollution, and habitat destruction, the wild resources of C. brachygnathus are under serious threat. However, up to date, there is still a lack of data on the genetic resources of C. brachygnathus in Huaihe River. In the present study, genetic diversity was analyzed in five C. brachygnathus populations from the Huaihe River basin of Anhui Province by microsatellite test (Fig. 1). The results showed that 10 microsatellite loci were highly polymorphic and polymorphism information content (PIC) was from 0.852 to 0.942. The five populations showed high genetic diversity, with the expected heterozygosity (He) from 0.879 to 0.903 and PIC from 0.851 to 0.881 (Table 1, 2). AMOVA analysis showed that most of the genetic variationa were found within C. brachygnathus population (97.88%), while only 2.12% was found among populations (Table 3). Genetic differentiation and genetic distance analysis showed that the overall differentiation level was low (Fst < 0.05), Fst between Huihe River and Yinghe River populations was lowest at 0.004 45, while that between Fengtai and Wangjiaba was the highest at 0.041 02. The genetic distance between Huihe River and Yinghe River population was the lowest at 0.160 9, while that between Fengtai and Wangjiaba population was the highest at 0.559 5 (Table 4, Fig. 2). The population genetic structure analysis based on Structure software indicated that the 5 populations belong to 3 genetic lineages (Fig. 3). In addition, the five C. brachygnathus populations might experience the bottleneck effect, especially in the Huaiyuan and Fengtai populations (Table 5). In conclusion, the wild resources still have high genetic diversity, according to the genetic structure of the five C. brachygnathus populations in the Huaihe River basin of Anhui Province, which we recommend to be one protection unit for protection and management. The results of the present study may enhance the understanding of C. brachygnathus populations in the Huaihe River, which will provide reference for the resource conservation and genetic improvement, and promote sustainable development and utilization of fish resources in the Huaihe River region.