A craniometric and molecular genetic investigation was conducted in Danish stoat (Mustela erminea) and weasel (Mustela nivalis) populations. Specimens used were collected over a wide time span (stoat: 1864–2002; weasel: 1863–1990) and from several geographical regions (Jutland peninsula and the two islands of Funen and Zealand). The study was made with a temporal and a spatial perspective, allowing the estimation of differences in genetic diversity and craniometrical trait means between geographical regions and through time with the use of ancient DNA techniques. Univariate statistics of 11 trait lengths did not reveal geographical differentiation in size and shape among the different regions for the stoat, but a geographical differentiation in shape was found for the weasel. There was evidence for reductions in skull size with the year of collection in male stoats, but not in females, which suggests that some selective pressures or environmental factors have affected male stoats to a greater extent than female stoats and the weasel. Relatively high values of heterozygosity were found in both the stoat and weasel, using microsatellite markers. The level of genetic variability of the stoat collected recently was compared with the level of genetic variability in the historical samples, demonstrating that the stoat has not suffered severe loss of genetic variability through the investigated period. A comparison of recent and historical genetic variability of the weasel was not possible because the ancient DNA extracted from the weasels was too degraded. Pairwise FST values and assignment tests showed small but significant genetic differentiation between the different geographical regions for both the stoat and weasel. No genetic differentiation between the recent and historical samples of the stoat was found. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 541–553.