We have amplified and sequenced 679 nucleotides of the mitochondrial DNA control-region in 45 Siberian (Capreolus pygargus) and European (C. capreolus) roe deer from two localities in Russia and seven in Italy. Average interspecific sequence divergence was 4.9%. Six different haplotypes were found in Siberian roe deer, and 14 haplotypes in Alpine European roe deer. A population of the endemic Italian subspecies C. c. italicus was monomorphic bearing a single haplotype with one unique nucleotide deletion and a fixed transversion. Phylogenetic relationships among haplotypes indicated that the two species were separated with 100% bootstrap support, and there were two distinct population clusters within each species. These clusters correspond to different geographical locations of the samples: Siberian roe deer were subdivided into west Siberia (Kurgan region) and east Siberia (Amur region), and European roe deer were subdivided into an eastern and a western Alpine group. Average sequence divergence among conspecific populations was 1.2%. Calibrations of evolutionary rates of the different domains of the control-region suggest that Siberian and European roe deer speciated about 2–3 million years ago, and haplotype diversity within species was generated during the last 150 000–370 000 years. Geographical structuring of sequence variability in roe deer allows us to identify historical and recent intraspecific population differences, including the effects of human disturbance. The genetic peculiarities of the endemic Italian subspecies C. c. italicus call for careful conservation of its surviving populations.