Topmouth culter (Culter alburnus) is an economically important fish species distributed in rivers, lakes and reservoirs throughout China. This study focused on the genetic diversity and genetic germplasm resources of C. alburnus populations. We developed a set of 14 polymorphic multi-nucleotide repeat microsatellites using specific locus amplification fragment sequencing (SLAF-seq). Based on microsatellites and mitochondrial DNA D-loop sequences analysis, we identified six C. alburnus populations that had a high level of genetic diversity. Among them, the Xingkai Lake (XKL) population had the lowest genetic diversity, so it might require strengthened protection measures. XKL and Danjiangkou Reservoir (DJKR) populations were relatively closely related, and genetically distant from the remaining four populations. These results shall help us effectively monitor the germplasm resources of C. alburnus and provide scientific guidance for its resource development, utilization and protection.