Demographic bottlenecks can result in a loss of genetic variation due to the bottleneck effect and subsequent genetic drift. The arctic fox population in Scandinavia went through a severe demographic bottleneck in the early 20th century, and is today classified as critically endangered. In this study, we investigated the pre-bottleneck genetic variation in Scandinavia and compared it to modern samples from Scandinavia and North Russia. Variation in the mtDNA control region and five microsatellite loci was examined through ancient DNA analysis on museum specimens. The microsatellite data from the museum specimens was further used to simulate the expected effect of the bottleneck. The arctic foxes in Scandinavia have lost approximately 25% of the microsatellite alleles and four out of seven mtDNA haplotypes. The results also suggest that the genetic differentiation between North Russia and Scandinavia has doubled over the last 100 years. However, the level of heterozygosity was significantly higher than expected from the simulations. This highlights both the advantage of using museum specimens and the importance of generating specific predictions in conservation genetics.