Dinaric–Balkan and Carpathian gray wolf (Canis lupus L., 1758) populations, although geographically separated, occupy closely situated areas in the territory of Serbia. We studied morphological variation in the skull to investigate whether the two populations are distinguishable despite the animals being very large and highly mobile and seeming to form one continuous population. The size and shape of the mandible and cranium in the lateral view were compared between sexes and populations using landmark-based geometric morphometrics. Significant differences between sexes and populations were found for size and shape of both structures. Males were larger than females in both populations, and Carpathian were larger than Dinaric–Balkan wolves. Principal component analysis revealed the cranium to be a highly discriminative skull structure. Zygomatic arches were located anteriorly in males and posteriorly in females. Dinaric–Balkan wolves had a more elevated snout and sagittal crest than Carpathian wolves. Difference between Dinaric–Balkan and Carpathian wolves in the cranial flexion was observed for the first time in a comparative study of wolf populations. Present-day separation of Dinaric–Balkan and Carpathian wolf populations could have originated from 2 different glacial refuges. We presume that the observed morphometric divergence between the populations is not only a relic of the last glacial maximum but was further strengthened and shaped by synergistic effects of environmental factors and social behavior (territoriality and a dispersal mechanism influenced by the natal habitat).