Abstract:Sequence analysis and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) technique were used to identify the species of Thunnus thynnus, T. alalunga, T. obesus and T. albacares. Genetic variation of 376 bp fragments in mitochondrial cytochrome b gene of 4 Thunnus species captured from northern Pacific Ocean, northern Atlantic Ocean and Indian Ocean was analyzed. The restriction enzymes and restriction fragment length pattern in each Thunnus species were obtained. It was found that the 376 bp fragment of cytochrome b gene in T. obesus showed the obvious divergence of 4.235% different from other 3 species. The highest interspecific similarity ranged 98.687～99.674% between T. thynnus and T. alalunga. PCR-RFLP by using 3 specific restriction enzymes Bsp1286, Hinc II and Rsa I could precisely and quickly identify the species of the 4 common Thunnus species. Furthermore, this technique successfully detected the species of 12 samples of commercial tuna fillets (sashimi).