Abstract We have identified a large number of microsatellites from Pseudosciaena crocea, an economically important fish species in China, by taking advantage of the Roche 454 sequencing technology. A total of 2535 simple sequence repeats (SSR) motifs were identified among which dinucleotides were the most frequent (59.61%), followed by tetra- (29.94%) and trinucleotides (7.06%). Among dinucleotide repeats, the most frequent repeats were AC motifs, accounting for 79.15% of the total. AAT (36.31%) and TCTA (39.66%) motifs were the most frequent of the tri- and tetranucleotide motifs, respectively. A total of 1068 microsatellite loci had flanking sequences suitable for setting primers for polymerase chain reactions (PCR). To verify the identified SSRs, 40 primer pairs were randomly selected for PCR. In total, 32 primer sets (80.00%) produced strong PCR products matching their expected sizes, and 27 (67.50%) were polymorphic. In an analysis of 34 wild individuals from the Zhangzhou Sea area (Fujian Province, China) with the 27 primer sets, the number of alleles per locus ranged from 5 to 11 and the mean allelic richness was 6.74. Linkage disequilibrium was not observed between any pair of loci, indicating that the markers were independent. A Hardy–Weinberg equilibrium test revealed significant deviation in 6 of the 27 microsatellite loci after sequential Bonferroni corrections. This microsatellite isolation method is cost- and time-effective in comparison to traditional approaches.