In order to develop methylation markers related to low salinity tolerance in Portunus trituberculatus, in this study, methylation sensitive high resolution melting curve method (MS-HRM) was applied to screen and validate methylation loci related to low salinity tolerance. A total of 8 methylation markers were developed from the transcriptome database of P. trituberculatus, six of which were significantly related to low salinity tolerance, exhibiting a significant demethylation or methylation pattern in the low salinity condition. They were located in V-type proton ATPase, H⁺/Cl^? exchange transporter, succinate dehydrogenase, NAD(P) transhydrogenase, phosphoglycolate phosphatase, NADH dehydrogenase genes, and these markers provided useful tools for marker-assisted breeding of low-salinity-tolerant strain of P. trituberculatus. The low salinity tolerance test and genotyping results of these 6 loci in wild populations and “Ningxiang-1” selective strain showed that the survival rate of “Ningxiang-1” was significantly higher than that of wild populations under low salinity conditions, and the demethylation of loci Pt-M1 and Pt-M2 in the selective breeding population was significantly increased to 100%, which may be beneficial to maintaining the balance of Na⁺ and Cl^? under low salinity conditions. This study provides basic data and favorable tools for genetic improvement of low salinity resistance in P. trituberculatus.