A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method had been developed for the detection of five billfish species Xiphias gladius, Makaira nigricans, M. indica, Istiophorus platypterus and Tetrapturus audax in raw, frozen and heat-treated meats. The primers L-CYTBF and H-CYTBF were designed in the mitochondrial cytochrome b (cytb) gene and the molecular weight of amplified fragment was 348 bp and amplified the fragment from processed billfish meats. The results obtained from the BsaJI, Cac8I and HpaII enzymes digestion could be used to distinguish the five billfish species in frozen and heat-treated meats. Using the PCR–RFLP method, species of 10 commercial samples including raw fish fillets, frozen fish meats and fried fish meats could be identified. It was determined that two commercial samples of billfish products were not made from billfish. The method is sensitive, rapid and valid to detect fraudulent billfish products substituted from cheaper fish.