Thaumetopoea wilkinsoni Tams, 1924 occurs in the southeast Mediterranean basin and infests pine and cedar stands. Our objective was to decipher the biogeography of the species and, in particular, to explore its evolutionary history on the island of Crete. We collected 135 individuals from 14 sites on Crete, Turkey, Samos and Rhodes. We sequenced one mitochondrial fragment (cytochrome c subunit I) and used 12 microsatellite loci for population genetic analyses. All results supported the deep divergence of Cretan populations from the neighbouring areas, with the differentiation between Crete and neighbouring regions being similar to the interspecific divergence between T. wilkinsoni and its sibling species, Thaumetopoea pityocampa (Denis and Schiffermüller, 1775). Both mitochondrial and microsatellite markers showed a clear longitudinal (east-west) differentiation of populations within Crete. Our results thus suggest that T. wilkinsoni crossed the Mid-Aegean Trench, became isolated on Crete and differentiated through vicariance after the end of the Messinian Salinity Crisis, favouring the emergence of an endemic lineage. There, Quaternary climatic oscillations coupled with geographical barriers to gene flow have given rise to the currently observed east-west differentiation of Cretan populations.