A rapid polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis was designed to examine genetic differentiation of some penaeid shrimps. Three hundred and fifty-six bp of cytochrome-b gene, a specific part of mitochondrial genome, was amplified with PCR to figure out differences between shrimp species Penaeus semisulcatus, Penaeus kerathurus, Parapenaeus longirostris, and Metapenaeus monoceros. PCR products were digested with different restriction endonucleases (DdeI, MboI, MboII, and MseI.). None of the tested enzymes, alone, was able to distinguish between the four shrimp species, but with the combination of the results of two digestions, all of the species could be differentiated. It was shown that PCR-RFLP method can be used to expose fraudulent substitutions of processed shrimps prior to marketing.