Over 500 unrelated individuals from the three major population groups in Bulgaria (Bulgarians, Bulgarian Turks and Roma) were analyzed for 17 highly polymorphic DNA loci (7 autosomal, one X-linked and 9 Y-chromosomal) to study the genetic structure of the Bulgarian population. Significant differences in both allele and genotype/haplotype frequencies were observed between three population groups. Bulgarian Turks showed to be the most heterogeneous group in Bulgaria with the highest variability in all studied loci. In seven of 17 investigated loci they demonstrated significant differences with Bulgarian allele frequencies. The exact test of sample differentiation between Bulgarians and Bulgarian Turks, based on Y-haplotype data, showed insignificant differences (P=0.13), while AMOVA analysis of Y-haplotypes revealed low, but significant Fst (0.027; P<0.00001). Network analysis of Y-chromosomal haplotypes demonstrated high level of haplotype sharing and clustering of some rare "adjacent" Bulgarian and Turkish haplotypes that could be due to admixture between Bulgarians and Bulgarian Turks. Bulgarian Roma can be clearly distinguished from Bulgarians and Bulgarian Turks on the basis of 15-13 of 17 analyzed loci (Y-chromosomal Fst=0.101 - 0.04, P<0.00001). Despite the observed strong founder "Vlax Roma" effect of Y-chromosome haplotype distribution in this group, the network analysis revealed several Y-haplotype clusters thus suggesting heterogeneous structure of this group. Y-chromosomal haplotype distributions in the three studied Bulgarian population groups were compared with the neighboring Balkan populations. Observed heterogeneity of Bulgarian population is important for genetic studies and forensic practice in Bulgaria.