The red deer ('Cervus elaphus') population in Denmark became almost extinct in recent historical times due to over-hunting. The species has subsequently recovered within remote areas, but non-Danish individuals have been introduced at several localities. To assess genetic structure, past demographic history, and the possibility of a still existing original stock, we analysed 349 specimens from 11 geographically separate areas and from three enclosed areas, genotyping 11 microsatellite loci. Moreover, an 826-bp fragment of the control region of the mitochondrial DNA was sequenced for 116 recent specimens and seven museum specimens. There was a significant difference in mean expected heterozygosity (HE) between the three enclosed areas and the 11 unenclosed areas. Significant departures from Hardy–Weinberg equilibrium were observed in the three enclosed areas and in nine of the unenclosed areas. The overall degree of genetic differentiation among all 14 areas was significant (FST = 0.09, P < 0.01), primarily because the mean pairwise FST for the three enclosed areas was significantly higher than that for the 11 unenclosed areas. A Bayesian clustering procedure detected three genetically distinct populations and indicated reduced gene flow between the enclosed and unenclosed areas. The individuals in the unenclosed areas show genotypic mixture, presumably as a result of gene flow among them. Markov Chain Monte Carlo simulations, based on the genealogical history of the microsatellite alleles, suggest a drastic decline in the effective population size of the enclosed areas some 188–474 years ago. Mitochondrial DNA analysis of the recent specimens showed seven haplotypes. Individuals from the enclosed Jægersborg Dyrehave contain haplotypes that occur all over Denmark and also are found in Western Europe. A close relationship between Scandinavian and Western European red deer is most likely. Only individuals from the unenclosed Lindenborg Estate and the enclosed Tofte Skov did not group with any other Danish individuals. As six of seven museum specimens had haplotypes also found in modern Danish samples, current population of red deer in Denmark is genetically close to the original Danish red deer.