Polyacrylamide gel electrophoresis was used to assess genetic variability and differentiation at 40 gene loci in 102 roe deer from five sampling sites in the province of Siena, central Italy. Eight loci proved to be polymorphic in at least one population. Averaged over populations, values of expected heterozygosity (mean He = 0.044, SD = 0.007) and the proportion of polymorphic loci (mean P = 0.135, SD = 0.028) were as high as those found for roe deer of southern and central Europe. In spite of a moderately high level of gene flow between sites (Nm = 4.7), the existence of some degree of genetic structuring in the whole population of Siena could be demonstrated. Demographic events (i.e., restocking, bottlenecks), rather than genetic isolation were explained to be responsible for the differences in allele frequencies found among populations. The allochthonous origin of roe deer living on Mount Amiata could be proved by genetic data. The results are discussed in the light of historical and demographic information on this species in the province.