We determined allelic variation at eleven microsatellite loci to study the effect of the Bosphorus, the Sea of Marmara, and the Dardanelles on the population genetic architecture of brown hares, Lepus europaeus, from Anatolia and Turkish Thrace in the southeastern most Balkans. The latter region was connected with Anatolia during periods of the late Pleistocene and the early Holocene (until ca. 8000 y BP) enabling natural gene flow. Following a recent mitochondrial (mt) DNA-based phylogeographic model that suggested unidirectional ancient gene flow from Anatolia to the SE Balkans, we expected lower genetic diversity in hares from Turkish Thrace than Anatolia. The Turkish Thrace population might have been established only relatively recently as natural northwestward expansion of a long existing Anatolian population. Alternatively, it might have received late Pleistocene or Holocene gene flow by hares from eastern Europe from north of the Black Sea, even after the formation of the marine barrier. This might have produced a notable genetic distinction between the hares from either side of the Bosphorus, the Sea of Marmara, and the Dardanelles. We found significantly lower allelic richness in hares from Turkish Thrace than Anatolia, very little genetic admixture in the Turkish Thrace population, and only one allele at low frequency in Turkish Thrace that was not observed in Anatolian hares. On the contrary, many alleles of Anatolian hares were not recovered in hares from Turkish Thrace. Nevertheless, genetic divergence (Fst, Cavalli-Sforza & Edwards distances, AMOVA) between the hares from either side of the current marine barrier was low (Fst values <10%). All findings accord to our initial hypothesis that the hares from Turkish Thrace represent merely a somewhat genetically depleted version of the Anatolian hares. Seemingly, no strong drift has occurred in the Turkish Thrace population since the break-up of the Holocene landbridge, and there is no indication of gene flow from other regions in (eastern) Europe into the Turkish Thrace population. Spatial genetic approaches and standard population genetics (BAPS, GENELAND, STRUCTURE, F-statistics, Cavalli-Sforza & Edwards distances) indicated significant spatial genetic differentiation of hares across Anatolia, but also on a very low level. Overall, our results are concordant with the latest mtDNA based model of ancient gene flow in hares only from Anatolia to the southeastern Balkans across the Pleistocene and early Holocene landbridge; this implies that most or all of the Balkans have been colonized by hares exclusively originating eventually from Anatolia. (C) 2018 Deutsche Gesellschaft fur Saugetierkunde. Published by Elsevier GmbH. All rights reserved.