In the Mediterranean, numerous and frequent bio-invasions by non-native species have occurred in recent decades. Among the reasons for this biological pollution there is the recent trend of global warming, which led to the extension of distribution area of tropical and/or thermophilic species. The present investigation describes analyses of 12S and 16S mt-rRNA gene fragments conducted on fresh muscle tissues of Tyrrhenian Sea fishes that confirm the morphological records of Fistularia commersonii, Sphoeroides pachygaster and Trachipterus trachipterus and provides a 12S mt-rRNA fragments database for future investigations on Remora osteochir, Tetragonurus cuvieri, Pomadasys incisus and Sudis hyalina. Among the species examined, Fistularia commersonii and Sphoeroides pachygaster are allochthonous species penetrated into the Mediterranean through Suez Canal (Red Sea) and Gibraltar Strait respectively. Pomadasys incisus is thermophilus species basically restricted to the southern parts of the Mediterranean. The others, Sudis hyalina, Tetragonurus cuvieri, Trachipterus trachipterus, and Remora osteochir are all autochthonous species of sporadic occurrence. Accordingly, the present work proposes the unequivocal non-native species discrimination by polymerase chain reaction that may be used as an index of biological pollution.