We analysed the population genetics of the brown hare (Lepus europaeus) in order to test the hypothesis that this species migrated into central Europe from a number of late glacial refugia, including some in Asia Minor. Thirty-three localities in Greece, Bulgaria, Italy, Croatia, Serbia, Poland, Switzerland, Austria, France, Germany, the Netherlands, Spain, the United Kingdom, Turkey and Israel. In total, 926 brown hares were analysed for mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism (RFLP) performed on polymerase chain reaction-amplified products spanning cytochrome b (cyt b)/control region (CR), cytochrome oxidase I (COI) and 12S-16S rRNA. In addition, sequence analysis of the mtDNA CR-I region was performed on 69 individuals, and the data were compared with 137 mtDNA CR-I sequences retrieved from GenBank. The 112 haplotypes detected were partitioned into five phylogeographically well-defined major haplogroups, namely the 'south-eastern European type haplogroup' (SEEh), 'Anatolian/Middle Eastern type haplogroup' (AMh), 'European type haplogroup, subgroup A' (EUh-A), 'European type haplogroup, subgroup B' (EUh-B) and 'Intermediate haplogroup' (INTERh). Sequence data retrieved from GenBank were consistent with the haplogroups determined in this study. In Bulgaria and north-eastern Greece numerous haplotypes of all five haplogroups were present, forming a large overlap zone. The mtDNA results allow us to infer post-glacial colonization of large parts of Europe from a late glacial/early Holocene source population in the central or south-central Balkans. The presence of Anatolian/Middle Eastern haplotypes in the large overlap zone in Bulgaria and north-eastern Greece reveals gene flow from Anatolia to Europe across the late Pleistocene Bosporus land-bridge. Although various restocking operations could be partly responsible for the presence of unexpected haplotypes in certain areas, we nevertheless trace a strong phylogeographic signal throughout all regions under study. Throughout Europe, mtDNA results indicate that brown hares are not separated into discernable phyletic groups.