Here we study genetic differentiation and changes over time in genetic variability in the rare pine marten Martes martes. Samples from three isolated geographic regions: Jutland and Sealand (Denmark) and southern Scania (southernmost Sweden), were genotyped by sequencing the hypervariable domain of the mitochondria control region. Both recent and museum samples were analysed in order to evaluate any temporal loss of genetic variability. Eight haplotypes were found. Two were main haplotypes shared by individuals from all three regions, and in all localities unique haplotypes were found. When comparing the data with previous haplotype analysis, our results suggest that at least three different haplotype groups exist in central and Northern Europe, with the samples from southern Scania being differentiated from samples previously analysed from central Sweden, and the genotypic data for Jutland and Sealand suggest a recent independent evolutionary history for the Danish pine marten.