Scylla paramamosain is a commercially important crab species in China. Here we reported 12 novel polymorphic microsatellite markers isolated in S. paramamosain using PCR-based isolation of microsatellite arrays (PIMA). The analysis of genetic variability was performed in a sample of 29 individuals. A total of 82 alleles were detected with an average of 6.8 alleles per locus. The number of alleles, polymorphism information content (PIC), observed and expected heterozygosity per locus ranged from three to 12, from 0.42 to 0.88, from 0.38 to 1.00 and from 0.48 to 0.91, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0042) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated in three closely related crab species S. tranquebarica, S. olivacea, and S. serrata. This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and evaluating the population genetic diversity of S. paramamosain.