In this study, single nucleotide polymorphism (SNP) were identified, confirmed and genotyped in the mud crab (Scylla paramamosain) using Tm-shift assay. High quality sequences (13, 311 bp long) were obtained by re-sequencing that contained 91 SNPs, with a density of one SNP every 146 bp. Of all 91 SNPs, 40 were successfully genotyped and characterized using 30 wild specimens by Tm-shift assay. The minor allele frequency per locus ranged from 0.017 to 0.500. The observed and expected heterozygosity, and polymorphism information content (PIC) ranged from 0.000 to 0.600, from 0.033 to 0.509, and from 0.033 to 0.375, respectively, with an average of 0.142, 0.239 and 0.198 per locus. Seventeen SNPs were significantly deviated from Hardy-Weinberg equilibrium. No significant linkage disequilibrium between pairs of loci was detected after sequential Bonferroni correction (P > 0.00125). Seventeen SNPs were related with known function genes. This study provided new molecular markers for investigation of population genetic diversity, construction of genetic linkage maps and molecular marker-assisted selection in this important crustacean species.