Water salinity condition is an important factor for artificial propagation of the swimming crab (Portunus trituberculatus). Low salinity (LS)-resistant strains are preferred by crab industries. Single nucleotide polymorphism (SNP), the third generation of molecular markers, can be utilized in the breeding of LS-resistant species of P. trituberculatus. Our earlier study identified 615 genes differentially expressed in low-salinity stress compared to the controls. Although thousands of SNP loci have been found, it is hard to identify a SNP marker in correlation with a desired trait. In this study, time-of-flight mass spectrometry (TOF-MS), as an efficient method to select SNPs for the tolerance of LS challenge, was utilized for SNP typing. Fifty gene segments were amplified based on comparative transcriptomics in our earlier study, a total of 18 511 bp DNA fragments were amplified, and eighty-five SNP markers were found. The frequency of the SNPs was estimated to be 0.46 per 100 base pairs of DNA sequences. The rate of the conversion mutation was 81%, while the transversion mutation was 19%. The mutation rate of the G/T (C/A), A/T and G/C was 26%, 12% and 7%, respectively. Eight SNP markers were found to significantly correlate with the adaption of low salinity. Of the eight SNP markers, three linked-SNPs were found in the cuticle proportion gene, and another three SNPs were found in three new genes, and the rest two were found in aquaporin gene and chloride channel gene. The development of these SNP markers found in our study could be primarily used for breeding LS-resistant strains of P. trituberculatus.