Mitochondrial DNA variability in the Polish Roma population has been studied by means of hypervariable segment I and II (HVS I and II) sequencing and restriction fragment-length polymorphism analysis of the mtDNA coding region. The mtDNA haplotypes detected in the Polish Roma fall into the common Eurasian mitochondrial haplogroups (H, U3, K, J1, X, I, W, and M*). The results of complete mtDNA sequencing clearly indicate that the Romani M*-lineage belongs to the Indian-specific haplogroup M5, which is characterized by three transitions in the coding region, at sites 12477, 3921 and 709. Molecular variance analysis inferred from mtDNA data reveals that genetic distances between the Roma groups are considerably larger than those between the surrounding European populations. Also, there are significant differences between the Bulgarian Roma (Balkan and Vlax groups) and West European Roma (Polish, Lithuanian and Spanish groups). Comparative analysis of mtDNA haplotypes in the Roma populations shows that different haplotypes appear to demonstrate impressive founder effects: M5 and H (16261–16304) in all Romani groups; U3, I and J1 in some Romani groups. Interestingly, haplogroup K (with HVS I motif 16224-16234-16311) found in the Polish Roma sample seems to be specific for Ashkenazi Jewish populations.