Black Carp (Mylopharyngodon piceus) is an important aquaculture species in China. Developing microsatellite DNA markers from genome of M. piceus will provide a tool for its genetic diversity analysis. In this study, two biotin probes (CA)10 and (GACA)6 were used to construct two microsatellite enrichment libraries of M. piceus by magnetic beads enrichment. A series of microsatellite primers was designed and their polymorphisms were tested in a population of M. piceus. Softwares GeneMapperv4.0, Cervus3.0 and PopGen32 were used to analyze their genetic diversity information. Microsatellite markers with high polymorphism and rich genetic diversity were screene. Through analysis, 849 sequences containing microsatellite loci were obtained from 993 positive clones. The repeat number of the repeating unit was then counted (Fig. 1). The statistics showed that most numbers of the repeats were between 5 and 21. The repeating units were classified according to the number of nucleotides in each repeating unit. The results showed that the number of dinucleotide microsatellite loci was the most, as high as 1 076. It accounted for 82.45% of the total. There were 217 trinucleotide type microsatellite loci, accounting for 16.63% of the total. In trinucleotide type, only 0.77% of all 39 microsatellite markers were designed and assessed in 36 individuals of black carp from Wujiang population. Results showed that 25 microsatellite loci showed polymorphism (Table 1). The mean number of alleles (Na) and effective number of alleles (Ne) were 7.08 and 3.526, respectively. The mean value of observed heterozygosity (Ho) and expected heterozygosity (He) were 0.602 and 0.619, respectively. The mean value of polymorphic information content was 0.568. Mp23, Mp27 and Mp35 were extremely significant deviating from Hardy-Weinberg equilibrium (P < 0.01) (Table 2). The microsatellite primers developed in this study will provide a tool and theoretical basis for evaluation and protection of germplasm resources, and genetic diversity analysis in M piceus.