The present study aimed to demonstrate the effectiveness of the 5s rDNA gene for the identification of commercially-valuable species of cephalopod belonging to the families Loliginidae and Ommastrephidae. Our results demonstrate distinct banding patterns in each of the six species sampled (Loligo surinamensis; Loligo sanpaulensis; Lolliguncula brevis; Sepiotheuthis sepioidea; Ornithoteuthis antillarum; Illex argentinus), as well as diagnostic traits at the genus and probably family levels. The results emphasize the efficiency of the 5s rDNA marker as a low-cost and rapid forensic technique, which not only permits the identification of species, but also differentiation of members of the Loliginidae and Ommastrephidae.