Two hundred and ninety-nine specimens of Atherina boyeri, collected from five Mediterranean sites (three from the open sea (Chioggia, Catania and Gaeta) and two from river mouths (the Birgi and S. Bartolomeo rivers) were analyzed in order to confirm previous genetic studies. Eigtheen loci were scored from 12 enzymatic systems, 3 of which were polymorphic: EST*, PGM* and PGI*. Mean Fst value (0.300, pFst and genetic distance revealed that samples from the river mouths (Birgi and S. Bartolomeo) were grouped separately from the open sea samples (Catania, Chioggia and Gaeta). These results are in agreement with the most recent classification, which considers A. boyeri as a complex of 3 different species.