The Pyrenean desman (Galemys pyrenaicus) is a small, semiaquatic mammal endemic to the Pyrenean Mountains and the northern half of the Iberian Peninsula where it lives in cold and well-oxygenated flowing mountain streams. This species is currently classified as vulnerable on the IUCN Red List and has been undergoing habitat loss and fragmentation for decades, inevitably impacting its distribution. A recent genetic study, based on mitochondrial and intronic sequences, showed that the genetic variability of the Pyrenean desman is very low in the Pyrenees. In this study, we investigated the potential existence of genetic structure and gene flow at a smaller scale using 24 polymorphic microsatellite loci. As the Pyrenean desman is a very elusive species, we supplemented our tissue sample collection with samples of feces collected in the French range of this species. We successfully identified 70 individuals based on 355 fecal samples. Bayesian analyses revealed 3 genetic and geographic clusters (1 eastern, 1 central, and 1 western, including 3 genetic subclusters), with origins tracing back only 200 years. These clusters were characterized by low levels of genetic diversity and high inbreeding coefficients. Although gene flow among clusters appeared to be limited, populations seem to have exchanged alleles recently. Therefore, connectivity between watersheds should be enhanced to maintain genetic diversity and potentially improve the long-term survival of the Pyrenean desman in France.