During the last century, the European polecat Mustela putorius populations in most of Europe declined and survived in fragmented patches, because of habitat alterations and direct persecution. To assess the genetic consequences of the demographic decline and to describe the spatial pattern of genetic diversity, 250 polecats sampled at seven localities from five European countries – Poland, Denmark (southern Denmark and northern Denmark), Spain, Belgium (eastern and western) and the Netherlands – were screened by means of nine microsatellite loci. Genetic diversity estimated by mean expected heterozygosity (HE) and allelic richness (AR) were moderately high within populations [range: 0.50 (northern Denmark) ≤HE≤0.64 (Poland) and 1.33≤AR≤7.80] as compared with other carnivores and mustelids. Bottleneck tests suggested that polecat populations in southern Denmark and Poland have declined recently and populations from northern Denmark and the Netherlands have expanded recently, whereas the remaining populations did not show any sign of demographic change. Recent demographic changes could suggest that some of the populations are still not in equilibrium, which could partly explain the relatively high genetic variability observed in polecat populations despite the drastic decline in population size observed in several European countries. A significant heterozygote deficiency [FIS=0.19; 0.01≤95% confidence interval (CI)≤0.32] suggests substructuring within the total European sample. Partitioning of the genetic variation among sampling locations (FST=0.14; 0.06≤95% CI≤0.23) and pairwise FST between localities (range: 0.01≤FST≤0.37) without any correlation with the geographic distances between localities were found, suggesting a recent divergence and a restriction of gene flow between populations and the action of genetic drift. An assignment test showed that the Polish and the northern Danish populations were the most unique, whereas the other populations were partially admixed. Factorial component analysis tests indicate a subdivision of the total sample into two distinct groups: one including the samples from Poland and the two Danish localities and the second group comprising the remaining localities investigated. The observed pattern of genetic differentiation is suggested to be due to two main routes of recolonization after the last glacial period. To compare the results obtained with microsatellite data, the most variable region of the mitochondrial DNA (d-loop) was sequenced and different phylogenetic reconstructions and genetic diversity analyses based on nucleotide (π) and haplotype diversity (h) measures within populations were performed using a subsample of populations. The lack of well-defined geographical structure, as well as the reduced level of mitochondrial DNA variability (π: 0.00274±0.00038; h: 0.876±0.028) that was found, has been previously reported in several studies on different carnivores and supports the hypothesis of post-glacial recolonization from southern or eastern refugees of Europe as suggested by the microsatellite data. Implications for conservation strategies of the polecat at the European level are discussed.