© 2018 Valbuena-Ureña et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The Pyrenees represent a natural laboratory for biogeographic, evolutionary and ecological research of mountain fauna as a result of the high variety of habitats and the profound effect of the glacial and interglacial periods. There is a paucity of studies providing a detailed insight into genetic processes and better knowledge on the patterns of genetic diversity and how they are maintained under high altitude conditions. This is of particular interest when considering the course of past climate conditions and glaciations in a species which is considered site tenacious, with long generation times. Here we analyzed the genetic patterns of diversity and structure of the endemic Pyrenean brook newt (Calotriton asper) along its distribution range, with special emphasis on the distinct habitat types (caves, streams, and lakes), and the altitudinal and geographical ranges, using a total set of 900 individuals from 44 different localities across the Pyrenean mountain range genotyped for 19 microsatellite loci. We found evidence for a negative longitudinal and positive altitudinal gradient of genetic diversity in C. asper populations. The fact that genetic diversity was markedly higher westwards is in accordance with other Pyrenean species. However, the impact of altitudinal gradient on the genetic diversity seems to differ from other species, and mostly from other amphibians. We found that lower altitudes can act as a barrier probably because the lowlands do not provide a suitable habitat for C. asper. Regarding the distinct habitat types, caves had significantly lower values of genetic diversity compared to streams or lakes. The mean FST value was relatively high (0.304) with maximum values as high as 0.771, suggesting a highly structured total population. Indeed, populations were grouped into five subclusters, the eastern populations (cluster 1) remained grouped into two subclusters and the central-western Pyrenees (cluster 2) into three subclusters. The increase of isolation with geographical distance is consistent with the population structure detected. In conclusion, C. asper seems to be adapted to high altitude mountain habitats, and its genetic diversity is higher in the western Pyrenees. In terms of conservation priority, we consider more relevant the populations that represent a reservoir of genetic diversity.