A method of authenticating anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) semipreserves (salt-cured and fillets in oil) has been developed by polymerase chain reaction (PCR) followed by sequence and restriction site analysis. The amplification of a fragment of the cytochrome b gene by universal primers produced a 376 base pairs (bp) fragment in all samples analyzed. Digestion of PCR products with XhoI, TaqI, AluI, and HinfI endonucleases yielded species-specific profiles distinguishing anchovy from gilt sardine. Therefore, the restriction length fragment polymorphism (RLFP) technique can be used to determine the species identity of anchovy and gilt sardine in semipreserves.