The aim of this study was to see if a simple, low-cost anatomical method could be used to identify genetic differences among different populations of pouting Trisopterus luscus. We based our study on 8 anatomical units of the pouting skeleton in 170 specimens from 4 populations (3 Atlantic, 1 Mediterranean). Significant variations in the relative size of the respiratory anatomical structures and other cranial regions were present. These differences are thought to be a consequence of the different environmental conditions to which the fish are exposed. This is most noticeable along the Mediterranean coasts of the Alborán Sea, where pouting show a significant increase in the size of skeletal structures involved in respiration, whereas the Atlantic populations showed no interpopulation variation, suggesting the existence of a genetic barrier between the Mediterranean and Atlantic populations. Further research is needed to identify possible environmental factors involved.