The Eurasian otter (Lutra lutra) suffered a dramatic population decline in southern Scandinavia in the twentieth century and was subsequently presumed extinct in the Danish archipelago. However, in the 1990s, evidence of a relict population on the island of Zealand was reported. The vulnerability of this small population may be exacerbated by seawater barriers preventing migration to and from the island. To assess whether the Danish Straits have and will present a barrier to future otter migration, we used geometric morphometrics to investigate population structuring and assess the level of differentiation of otters across southern Scandinavia. Utilizing museum collections, we analysed 137 otter skulls from Zealand, Jutland, and southern Sweden. Statistical analyses showed highly significant shape differences between all three localities, likely reflecting migration between the three areas has historically been low. High jack-knife reclassification success rates support divergence among the areas, and directionalities of the vectors describing regional shape differences indicate that differences do not reflect isolation-by-distance.