Representatives of the genus Salamandra occur in Europe, Northern Africa and the Near East. Many local variants are known but species and subspecies status of these is still a matter of dispute. We have analysed samples from locations covering the whole expansion range of Salamandra by sequence analysis of mitochondrial D-loop regions. In addition, we have calibrated the rate of divergence of the D-loop on the basis of geologically dated splits of the closely related genus Euproctus. Phylogenetic analysis of the sequences suggests that six major monophyletic groups exist (S. salamandra, S. algira, S. infraimmaculata, S. corsica, S. atra and S. lanzai) which have split between 5 and 13 million years ago (Ma). We find that each of the Salamandra species occupies a distinct geographical area, with the exception of S. salamandra. This species occurs all over Europe from Spain to Greece, suggesting that it was the only species that has recolonized Central Europe after the last glaciation. The occurrence of specific east and west European haplotypes, as well as allozyme alleles in the S. salamandra populations suggests that this recolonization has started from at least two source populations, possibly originating in the Iberian peninsula and the Balkans. Two subpopulations of S. salamandra were found that are genetically very distinct from the other populations. One lives in northern Spain (S. s. bernardezi) and one in southern Italy (S. s. gigliolii). Surprisingly, the mitochondrial lineages of these subpopulations group closer together than the remainder S. salamandra lineages. We suggest that these populations are remnants of a large homogeneous population that had colonized Central Europe in a previous interglacial period, approximately 500 000 years ago. Animals from these populations were apparently not successful in later recolonizations. Still, they have maintained their separate genetic identity in their areas, although they are not separated by geographical barriers from very closely related neighbouring populations.