Correct identification of fish species including their origin requires a compilation of recognized material to verify the conformity of the product with the labelling requirement. Sardine is among the internationally disputed example of species identification conflicts, with the lack of genetic reference material at the Southern part of the Mediterranean. Therefore a method was developed to discriminate between Tunisian small pelagic fish: Sardina pilchardus, Sardinella aurita and Engraulis encrasicolus.