A double-DNA approach was developed to discriminate the three Trachurus species that abide in European waters: T. trachurus, T. mediterraneus, and T. picturatus. The analysis aimed at both mitochondrial and nuclear loci. Polymerase Chain Reaction (PCR) amplification of the cytochrome b gene of mtDNA was followed by restriction analysis with three species-specific enzymes: NlaIII, NciI, and BsmAI. Digestion with these endonucleases yielded species-specific electrophoretic profiles. The universality of the results was verified by screening a large number of individuals from 12 geographical regions covering most of the distribution of the species. Additionally, the nuclear multicopy 5S rRNA gene was selected as an alternative candidate for the discrimination of the three Trachurusspecies. A simple agarose gel electrophoretic analysis of the amplicons proved to be capable of leading to unambiguous identification of the three Trachurus species. Thus, the double-DNA methodology presented here allows the accurate discrimination of Trachurus fish species and the detection of commercial fraud.