The garden dormouse, Eliomys quercinus (Rodentia, Gliridae), displays a surprisingly high karyotypic diversity, with the number of chromosomes varying between 2N = 48 and 2N = 54. We aimed to assess whether the karyotypic diversity is congruent with the mitochondrial differentiation of the populations; improve our understanding of the taxonomic relationships between garden dormouse populations based on both chromosomal and mitochondrial information; and establish the phylogeographic history of the species and the time of differentiation of mitochondrial lineages of E. quercinus and E. melanurus. For this purpose we sequenced the mitochondrial cytochrome-b (Cytb) gene for 62 E. quercinus and 4 E. melanurus from 27 localities representing all the known chromosomal races of the genus Eliomys in the western Palearctic region. Our results 1st showed that populations of E. quercinus are separated into 4 evolutionarily significant units partially congruent with the chromosomal races and corresponding to Iberian (2N = 48), Italian (2N = 48 or 50), western European (2N = 48, 49, or 50), and Alpine (2N = 52 or 54) mitochondrial lineages or clades. The existence of hybrid individuals between chromosomal races and the presence of several chromosomal races within each mitochondrial lineage both indicate that gene flow persists between chromosomal races. Second, we estimated that the major mitochondrial lineages differentiated from each other around 4.2 ± SD 1 million years ago, thus predating the Quaternary glaciations. Third, E. quercinus displayed a higher haplotypic variability in the Mediterranean peninsula than in the northwestern European populations. However, E. quercinus did not show a pattern of postglacial recolonization of northwestern Europe from Iberian or Italian populations. Our results also suggest that additional, unexpected refuge regions around the Alps exist for the species. Such information will be useful for deciphering the priorities for the protection of E. quercinus, which is listed as “Near Threatened” on the International Union for the Conservation of Nature Red List of Threatened Species and is protected by Appendix III of the Bern Convention.