The three-spot swimming crab (Portunus sanguinolentus) is one of the most important large size economic crabs in China. In this study, we first isolated and characterized a set of 84 SNP loci in P. sanguinolentus. PCR products from ten pairs of primers were sequenced and a total of 3181 bp high-quality DNA sequences were obtained. After genotyping, 84 polymorphic SNP loci were identified. The average frequency of SNP loci was one locus per 38 base pairs in P. sanguinolentus genome. Of these 84 SNP loci, each had bi-alleles with the minor allele frequency ranging from 0.0167 to 0.5000. The observed heterozygosity varied from 0.0333 to 0.7143, while the expected heterozygosity ranged from 0.0333 to 0.5085 per locus. 51 loci showed low variation (H_O ≤ 0.3) and 14 SNP loci showed high variation (H_O ≥ 0.5). Among 84 SNP loci, 11 loci showed significant deviation from Hardy–Weinberg equilibrium. The developed SNP markers herein will provide valuable information for elucidating population genetic diversity, population dynamics, and conservation genetics of this germplasm resource and other related crab species.