Megalobrama pellegrini is one of the economically important freshwater fish in China. Here, we developed 29 polymorphic microsatellite loci of M. pellegrini. The number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (HO), expected heterozygosity (HE) and polymorphic information content (PIC) ranged from 3 to 11 (mean±SD 5.4828±1.9571), 2.8708 to 9.6257 (mean±SD 5.0865±1.6681), 0.4333 to 0.9333 (mean±SD 0.7874±0.1213), 0.6627 to 0.9113 (mean±SD 0.7946±0.0751) and 0.5785 to 0.8868 (mean±SD 0.7439±0.0950), respectively. Cross-species amplification was successful at most loci for related species such as M. amblycephala, M. hoffmanni, M. skolkovii and Parabramis pekinensis. The transferability rate of the 29 polymorphic microsatellite markers in M. amblycephala, M. hoffmanni, M. skolkovii and P. pekinensis were 96.55%, 86.21%, 86.21% and 75.86%, respectively. These polymorphic microsatellites are not only useful in genetic study and conservation of M. pellegrini, but also an effective tool for identifying the related species. We could use 5 microsatellite markers (HHF-63, HHF-104, HHF-113, HHF-148, HHF-163) to distinguish the 5 species.