Population structure and barriers to gene flow are important components for understanding the evolutionary history of a species. Here we study population structure and differentiation in the western capercaillie (Aves: Phasianidae) along the Carpathian Mountains. Further, we compared the levels of population differentiation among capercaillie from the Carpathian Mountains, Balkans (Bulgaria) and the boreal forest (Russia and Sweden) in order to reveal past and current processes which may influence population structure. Tissue samples, non-invasive faeces and feathers and toe pads from museum specimens were used for genetic analyses of mitochondrial (mtDNA) sequences and allelic variation at nine nuclear DNA (nDNA) microsatellite loci. Analyses of mtDNA sequences revealed a southern subclade within the northern clade. Within the northern clade, microsatellite data distinguished two groups: (1) Western Carpathian populations; and (2) Eastern Carpathian and boreal forest populations. Bulgarian populations constituted a third cluster corresponding to the southern phylogenetic subclade. The Western Carpathian populations showed a heterozygote deficiency. The analyses indicate that the abundant Eastern Carpathian populations share alleles with populations from the boreal forest suggesting a common origin of these populations since the last glacial period. On the other hand, the Western Carpathian populations have been isolated over a long period with only a few migrants from the east, thereby becoming differentiated from the eastern and northern populations. The southern populations have been isolated from the northern populations since the last glacial maximum. The molecular analyses did not support the currently recognised taxonomy at the subspecies level.