To obtain SNP mutations of mitochondrial genome in Portunus trituberculatus, a total of 179 primer pairs were designed, nearly covering the whole mitochondrial genome in this study. Of the 179 primer pairs, 89 got the expected specific amplification fragments by regular PCR. Then, 22 of 89 amplification fragments were found to contain the SNPs, by testing of HRM technology (High resolution melting curve). Though sequencing, 24 SNPs were obtained in the 22 amplification fragments. The results by statistical analyzing demonstrated that SNP frequency in mitochondrial genome of P. trituberculatus was 0.15/100 bp, in which the conversion mutation rate was 79.1%, 16.6% for transvesion mutation rate, 79.1% for mutation rate of C to T (or G to A), 8.3% for the mutation rate of G to T (or C to A), and 4.16 % for the mutation rate of G to C and A to T, respectively. Thus, the mutation of C to T (containing the type of G to A) is the most common mutation type. Additionally, the distribution of 24 SNP mutation loci was not even in the whole mitochondrial genome. The number of SNPs was the greatest in COX1 gene region, and the second is in D-LOOP and ND1 genes. However, no SNPs were found in some regions, such as tRNA, 12S rRNA. Our findings will provide an analysis tool that may help further researches in genetic diversity of germplasm resources and enhancement in P. trituberculatus.