The present study investigated the natural recovery of the Eurasian otter (Lutra lutra) in France. The otter was widely distributed in France at the dawn of the 20th century, but then its range considerably shrank and became highly fragmented until the early 1970s, just before it was legally protected. However, for more than 25 years, the otter has been reconquering several parts of its original range and is now considered to be in expansion in France. We investigated the genetic differentiation and diversity of several populations from western and central France and northern Spain to gain insight into the recolonisation dynamics of this elusive species. The present study, based on the use of 14 microsatellite markers, revealed that otter populations seem to be split into five distinct groups. The distribution of samples in those five clusters was closely correlated with suspected refugia where the otter probably survived during the 20th century. Admixture was observed between genetic lineages, possibly enhancing their genetic diversity and thus increasing the recolonisation dynamics of these populations. This phenomenon resembles the genetic pattern noted in many invasive exotic species derived from multiple sources and introduction events. Finally, a demographic approach revealed the probable link between historical human pressure and otter population fragmentation patterns.