The grey wolf ( Canis lupus ) is one of the most widely distributed mammals in which a variety of distinct populations have been described. However, given their currently fragmented distribution and recent history of human-induced population decline, little is known about the events that led to their differentiation. Based on the analysis of whole canid genomes, we examined the divergence times between Southern European wolf populations and their ancient demographic history. We found that all present-day Eurasian wolves share a common ancestor ca 36 000 years ago, supporting the hypothesis that all extant wolves derive from a single population that subsequently expanded after the Last Glacial Maximum. We also estimated that the currently isolated European populations of the Iberian Peninsula, Italy and the Dinarics-Balkans diverged very closely in time, ca 10 500 years ago, and maintained negligible gene flow ever since. This indicates that the current genetic and morphological distinctiveness of Iberian and Italian wolves can be attributed to their isolation dating back to the end of the Pleistocene, predating the recent human-induced extinction of wolves in Central Europe by several millennia.