In this study, microsatellite markers were developed from the BAC-end sequences and used to analyze the genetic structure and genetic differentiation in two populations in Zhikong scallop (Chlamys farreri). A total of 3 374 microsatellites were identified from the 17 477 BAC-end sequences (BESs), of which tetra-nucleotide motifs were most abundant (26.6%), followed by penta-nucleotide motifs (17.7%) and hexa-nucleotide motifs were the least (12.0%). 77.3% SSRs were successfully amplified(99/128), and 43 SSRs(33.6%) were polymorphic in the parentage of mapping population. In order to apply these BES-SSR markers, 14 polymorphic SSRs were chosen to amplify and then analyze the genetic diversity in Dalian population and Qingdao population. A total of 395 alleles were obtained at the fourteen microsatellite markers and the number of alleles in each locus ranged from 8 to 38 in the two populations. The average number of alleles (Na) was 18.928 6 and 26.214 3 respectively. The average effective number of alleles (Ne) was 11.750 5 and 17.089 1 respectively. The mean observed heterozygosity (Ho) was 0.510 0 and 0.420 4. The mean expected heterozygosity (He) was 0.915 6 and 0.945 0. The data suggested both populations have high genetic diversities. The mean polymorphic information content (PIC) was 0.894 0 and 0.930 2, which were both greater than 0.5, indicating the fourteen loci were highly polymorphic. The unbiased genetic identity index was 0.487 9, and the genetic distance was 0.717 7. The coefficient of gene differentiation (FST) and gene flow (Nm) between two populations were 0.024 3 and 10.017 9 respectively. Low genetic differentiation was observed between the two populations, and the variance mainly came from individual difference. Significant deviation was detected by Hardy-Weinberg equilibrium test. There was heterozygote deficiency at all loci. The results showed BAC-end sequences were an effective resource for development of SSR markers for genetic and genomic researches.