At the dawn of the 20th century, the Eurasian otter (Lutra lutra) was widely distributed in France. However, due to intensive trapping and poaching for its meat and fur, the distribution range of the Eurasian otter got highly fragmented. Since the otter received legal protection in the seventies, a recolonization of its former range from relictual refugia has taken place. The present study therefore aims at evaluating the natural recovery of the otter in France through the investigation of the genetic differentiation and diversity of several otter populations originating from Western and Central France, and Northern Spain (Navarra). Assessing the genetic diversity of endangered species is indeed of major concern in the field of conservation biology since low genetic diversity and inbreeding depression may lead to reduced fitness and lower the adaptative potential of individuals to environmental changes. To investigate the genetic variation within and among the otter populations, STRUCTURE analyses were performed on 14 polymorphic microsatellite markers and almost 6,000 SNPs genotypes. These analyses identified five distinct groups, which appear to coincide with the respective suspected refugia. Indeed, some remnant populations were suggested to have survived along the Atlantic coast and in the Massif Central. Inference on population history (DIYABC) allowed to confirm the probable link between ancient human pressures and populations fragmentation, with the progressive fragmentation over time into several distinct clusters. Recent secondary contact and admixture appeared to have occurred between the genetic clusters, enhancing their genetic diversity and potentially increasing their recolonization dynamics. These regions could be considered as the strongholds where the species survived.