During the past 50 years the number of badgers (Meles meles) in Denmark has declined by c. 50%. To assess the genetic consequences of the demographic decline, six DNA-microsatellite loci were used to analyse 139 badger tissue-samples, which were collected in 1995–98 from three zones (1, 2 and 3) in Jutland (Denmark). Results from contemporary samples were compared to data obtained from DNA extracted from teeth belonging to 39 badgers collected from zones 1 and 2 in 1957–66. The microsatellites showed a low to moderate polymorphism, and the within area genetic diversity (HE) was relatively low (0.308≤HE≤0.441) as compared to other carnivores and mustelids. Genetic differentiation among the three recent sampling zones was significant (FST=0.072, P<0.0001). The FST values between the two recent (FST=0.092, P<0.0001) and the two historical samples (zones 1 and 2; FST=0.073, P<0.0001) were significant and similar. The HE and FST of the recent and historical zones were not significantly different (P>0.05). Despite the drastic recent decline, Danish badgers did not show apparent signs of genetic bottlenecks in both recent and historical samples. The use of molecular techniques which allowed us to work with small amounts of degraded DNA extracted from old teeth, allowed us to exclude the hypothesis that genetic differentiation between zones 1 and 2 was owing to a barrier, which has appeared in the last 50 years. Furthermore, the low genetic variability and therefore the relatively high genetic differentiation shown by Danish badgers were also shown not to be the result of the recent drastic population decline.