The development of reliable and specific analytical methods for the simultaneous detection and quantification of different crab species in food is an urgent need for seafood industries, restaurants and quality control laboratories. There is a growing need for rapid, reliable, and reproducible test to verify species in commercial fish and seafood products. In international trade and global seafood consumption, along with fluctuations in the supply and demand of different fish and seafood species have resulted in intentional product mislabeling. The effects of species substitution are far-reaching and include economic fraud, health hazards, and illegal trade of protected species. Portunid crabs of the genus, Portunus viz. P. pelagicus and P. sanguinolentus and the genus, Scylla viz. S. serrata and genus, Charybdis viz. C. natator are economically important species in India. An ongoing problem in the crab industry is mislabeling due to the difficulty of species identification after processing. There is a chance of food substitution with the cheaper crabmeat for the more expensive ones by manufacturers. Blue swimming crab (Portunus pelagicus) is an economically important species fetching more demand in export market in USA and Europe due to its savory flavour and high nutritional value. There has been reports of possible substitution of meat from three spot swimming crab (Portunus sanguinolentus) that is of low commercial value with blue swimming crab (Portunus pelagicus), which is of high value. To overcome this commercial crab meat fraud, a DNA-based multiplex PCR assay was developed for rapid detection of crab meat product. Species-specific primer sets were designed based on the nucleotide variation in mt cyt b and mt 16S rRNA regions for P. pelagicus and P. sanguinolentus, respectively. The primer set specifically amplified fragments of 298 bp in P. pelagicus, and 16S rRNA fragment of 383 bp in P. sanguinolentus. The MPCR assay was specific and rapid, as it did not amplify the DNA of other crab species tested and detected the species specific crab within 2 h. The MPCR assay was also detected the species of the crab meat that has undergone different processing treatments like freezing, cooking and frying. The Real Time PCR (qPCR) assay based on SYBR Green was developed for the quantification of mixed meat of crab species belonging to P. pelagicus and P. sanguinolentus. The developed qPCR assay was sensitive and rapid, as it detected 0.0003 pg to 0.024 ng of genomic DNA within 150 min. A sharp defined melting curve with narrow peak was found indicating that there was no primer dimers in the amplicons and the efficiency of amplification was excellent. The developed MPCR and qPCR assays was also validated with the commercial pasteurized crab meat products obtained from five crab processing industries located in the Thoothukudi region of Tamil Nadu in the in-house and inter-house laboratories, and found efficient and reproducible. The developed MPCR and qPCR assays could be used by the Food Regulatory Authorities for authenticating Portunus pelagicus meat.