The four species of the Engraulidae family: European anchovy (Engraulis encrasicolus), Californian anchovy (Engraulis mordax), Peruvian anchoveta (Engraulis ringens), and Japanese anchovy (Engraulis japonicus) studied in this work are very similar morphologically, and it is very difficult to distinguish between them, especially when frozen or processed. We have used the 5S rDNA as a molecular marker to discriminate these four species and used specific primers designed for each species in the nontranscribed spacers (NTS) of these genes. Multiplex PCR was performed with three pairs of primers, and three different sizes were obtained: 597 bp E. encrasicolus, 598 bp E. japonicus, 380 bp E. mordax, and 250 bp E. ringens. For the species E. encrasicolus and E. japonicus, PCR-RFLP was used as an additional technique to distinguish between them because their NTS sequences showed considerable similarity.
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