The polymerase chain reaction (PCR) technique was employed to obtain a 464bp amplicon from the mitochondrial cytochrome b gene from gadoid species to study its ability to differentiate them. The sequences of this fragment from 16 species were analysed using a genetic distance method, and polymorphic sites were determined. The fragment was shown to be moderately polymorphic (151 sites), and this permitted the differentiation of most of the species. A phylogenetic tree construction using Tamura-Nei distances was employed to allow the identification of Gadidae species, each species resulted in a well-differentiated clade, with the exception of Gadus ogac and Gadus macrocephalus, which could not be differentiated. Based on the sequences obtained, three restriction enzymes, Dde I, Hinc II and Nla III, were selected to provide specific restriction profiles, which allowed the differentiation of 15 species of gadoids in a faster and less expensive way than sequencing. The PCR-restriction fragment length polymorphism methodology was also tested using commercial samples.