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A spatio-temporal study of genetic variation in the Danish pine marten ( Martes martes) populations from the Jutland peninsula and from the island of Sealand was performed using 11 microsatellite markers. Samples obtained from 1892 to 2003 were subdivided into historical (prior to 1970) and recent (from 1970) groups. As compared with the historical samples, there was a significant loss of genetic variation in the recent Jutland population, but not in Sealand. Effective population sizes were estimated using Bayesian-based software (TMVP). Historical effective population sizes were 5897 (90% highest probability density, HPD, limits: 1502–6849) in Jutland and 1300 (90% HPD limits: 224–5929) in Sealand, whereas recent effective population sizes were 14.7 (90% HPD limits: 10.9–23.5) in Jutland and 802 (90% HPD limits: 51.8–5510) in Sealand. Significant genetic differentiation ( FST) was found between the two historical samples, between the two recent samples, and between the historical and the recent sample in Jutland; whereas the FST value between the historical and the recent sample in Sealand was not significant. The significant genetic differentiation between the historical and the recent samples indicates changes in the genetic compositions over time, and the higher FST values between the two recent samples, as compared with the two historical samples, indicates that the populations in Sealand and Jutland have drifted apart within a short time span. No deviation from Hardy–Weinberg equilibrium was found within populations, indicating no further substructuring. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 457–464. [ABSTRACT FROM AUTHOR]