BACKGROUND: Species with strict ecological requirements may provide new insights into the forces that shaped the geographic variation of genetic diversity. The Pyrenean desman, Galemys pyrenaicus, is a small semi-aquatic mammal that inhabits clean streams of the northern half of the Iberian Peninsula and is endangered in most of its geographic range, but its genetic structure is currently unknown. While the stringent ecological demands derived from its aquatic habitat might have caused a partition of the genetic diversity among river basins, Pleistocene glaciations would have generated a genetic pattern related to glacial refugia. RESULTS: To study the relative importance of historical and ecological factors in the genetic structure of G. pyrenaicus, we used mitochondrial and intronic sequences of specimens covering most of the species range. We show, first, that the Pyrenean desman has very low levels of genetic diversity compared to other mammals. In addition, phylogenetic and dating analyses of the mitochondrial sequences reveal a strong phylogeographic structure of a Middle Pleistocene origin, suggesting that the main lineages arose during periods of glacial isolation. Furthermore, both the spatial distribution of nuclear and mitochondrial diversity and the results of species distribution modeling suggest the existence of a major glacial refugium in the northwestern part of the Iberian Peninsula. Finally, the main mitochondrial lineages show a striking parapatric distribution without any apparent exchange of mitochondrial haplotypes between the lineages that came into secondary contact (although with certain permeability to nuclear genes), indicating incomplete mixing after the post-glacial recolonization. On the other hand, when we analyzed the partition of the genetic diversity among river basins, the Pyrenean desman showed a lower than expected genetic differentiation among main rivers. CONCLUSIONS: The analysis of mitochondrial and intronic markers in G. pyrenaicus showed the predominant effects of Pleistocene glaciations on the genetic structure of this species, while the distribution of the genetic diversity was not greatly influenced by the main river systems. These results and, particularly, the discovery of a marked phylogeographic structure, may have important implications for the conservation of the Pyrenean desman.