A DNA methodology was developed to discriminate fry of six Mugilidae species found in the Mediterranean, namely Mugil cephalus, Mugil so‐iuy, Chelon labrosus, Liza aurata, Liza ramada and Liza saliens. Polymerase chain reaction (PCR) amplification of the 5S rDNA gene was used for the identification of the above six species. PCR products of two species showed different patterns on EtBr‐stained agarose gels; M. so‐iuy gave a pattern of three bands, while L. saliens gave a pattern of one band. Mugil cephalus, C. labrosus, L. aurata and L. ramada gave a pattern of two bands. Subsequent sequencing analyses revealed unique haplotypes for each of the remaining four species. This is a genetic technique that could be applied in hatcheries, for identification of fry mullet's species. Furthermore, 5S rDNA sequences of each of five of the studied species (i.e. M. cephalus, C. labrosus, L. aurata, L. ramada and L. saliens), as well as a sequence of Oedalechilus labeo that belongs to the Mugilidae family, were aligned and used for the examination of the phylogenetic relationships among them. Phylogenetic trees produced in our study are in general agreement with those presented in the literature. This is the first study to use a nuclear marker to elucidate the phylogeny of Mugilidae species.