During the twentieth century otter (Lutra lutra) populations in central and western Europe declined and became fragmented because of habitat alterations, chemical pollution and direct persecution. In this study we used microsatellites to describe spatial patterns of genetic diversity and subdivision in otters from eight populations in Europe. Genetic diversity was moderately high within populations (He = 0.45–0.77), and significantly partitioned among locations (FST= 0.17; RST= 0.16; P < 0.001). A Bayesian cluster analysis of multilocus genotypes assigned individuals to seven genetically distinct groups, which were partly concordant with the geographical origin of the samples. An assignment test of the individuals to the populations showed that the Danish and, to a lesser extent, the Spanish populations were unique and distinct, whereas the other populations were partially admixed. Inference of past demographic fluctuations from coalescent analysis suggested that otter populations probably declined several thousand years ago, with the exception of the Irish population for which no such decline could be detected. No genetic evidence for recent bottlenecks was found. The historical decline could stem from post-glacial founding events and recolonization of northern Europe after the last glacial maximum, or from more recent dry stages in the early Holocene in central Europe. On the basis of these results, we recommend that recovery plans should promote the expansion of existing natural populations through improvements of river and wetland habitats.