Genetic diversity is of paramount importance for individual fitness and evolutionary potential of populations. For conservation planning it is crucial to know how genetically diverse a species is and what factors may explain variation of genetic diversity among populations. Our aim was to evaluate the effects of landscape history, ecological isolation, and local population size on allelic richness of local populations in European ground squirrels (Spermophilus citellus). We genotyped 144 individuals from nine local populations collected in two neighbouring regions with decades of different landscape history. We assessed allelic richness, ecological isolation and local population size by eleven polymorphic microsatellites, the isolation index of Rodríguez and Delibes, and standardised counts of burrows openings, respectively. Statistical models indicated a strong effect of landscape history on allelic richness of local populations. Ecological isolation of local populations apparently played only a marginal role, and local population size was an unimportant factor. Our modelling results highlight the dominant role of landscape history for the genetic diversity of S. citellus. The strong landscape history effect encountered presently includes a different region-specific socio-economic development due to distinct agricultural systems in the two regions, especially after World War II. Levels of ecological isolation of local populations have diverged in an extent too small to explain variation of local allelic richness. The lack of a significant effect of local population size suggests that census sizes of the populations studied are all not critically low. Moreover, census and estimated effective population sizes were not closely related. Establishing corridors or translocating S. citellus in the species’ historical range should be encouraged to promote gene flow between local populations and counteract the loss of genetic diversity by drift, provided that no conflicting factors (ecological, epidemiological, etc.) exist.