The DNA analysis is the best approach to authenticate species in seafood products and to unveil frauds based on species substitution. In this study, a molecular strategy coupling Cytochrome Oxidase I (COI) DNA barcoding with the consolidated methodology of Restriction Fragment Length Polymorphisms (RFLPs), named COIBar-RFLP, was applied for searching pattern of restriction enzyme digestion, useful to discriminate seven different fish species (juveniles of and sold in Italy as "bianchetto" and sold as "rossetto"; icefish ; European perch, and the Nile Perch, striped catfish, ). A total of 30 fresh and frozen samples were processed for DNA barcoding, analyzed against a barcode library of COI sequences retrieved from GenBank, and validated for COIBar-RFLP analysis. Cases of misdescription were detected: 3 samples labeled as "bianchetto" were substituted by (2 samples) and (1 sample); 3 samples labeled as "persico reale" ( ) were substituted by and All species were simultaneously discriminated through the restriction pattern obtained with I enzyme. The results highlighted that the COIBar-RFLP could be an effective tool to authenticate fish in seafood products by responding to the emerging interest in molecular identification technologies.