The aim of the study was to use hybrid populations as well as island populations of the European brown hare (Lepus europaeus) to explore the effect of evolutionary events, such as the post-deglaciation translocations, spontaneous and human-mediated, local adaptation and the genetic drift in the shaping of the phylogeographic patterns of the species. For this purpose, we used molecular markers, both nuclear and mitochondrial, that are indicative for local adaptation as well as neutral markers to elucidate the patterns of population differentiation based on geographic isolation and the clade of origin. To broaden our analysis, we included data from our previous studies concerning mainland populations, to explore the genetic differentiation in the base of the geographic origin (mainland/island) of the populations.Our results suggest that local adaptation shapes the differentiation in both genomes, favoring specific alleles in nuclear genes (e.g. DQA) or haplotypes in mtDNA (e.g. Control Region, CR). mtDNA variation was found to be in a higher level and was able to give a phylogeographic signal for the populations. Furthermore, the degree of variation was influenced not only by the geographic origin, but also by the clade of origin, since specific island populations of Anatolian origin showed a greater degree of variation compared to specific mainland populations of the European clade. Concerning the hybrid population, we confirmed the existence of both clades in the territory and we provided a possible explanation for the lack of introgression between the clades.Our results indicate that the Quaternary's climatic oscillations played a major role in the shaping of the phylogeographic patterns of the species, by isolating populations in the distinct refugia, where they adapted and differentiate in allopatry, leading to genome incompatibilities observed nowadays.