The variation at 28 Y-chromosome biallelic markers was analysed in 256 males (90 Croats, 81 Serbs and 85 Bosniacs) from Bosnia-Herzegovina. An important shared feature between the three ethnic groups is the high frequency of the “Palaeolithic” European-specific haplogroup (Hg) I, a likely signature of a Balkan population re-expansion after the Last Glacial Maximum. This haplogroup is almost completely represented by the sub-haplogroup I-P37 whose frequency is, however, higher in the Croats (∼71%) than in Bosniacs (∼44%) and Serbs (∼31%). Other rather frequent haplogroups are E (∼15%) and J (∼7%), which are considered to have arrived from the Middle East in Neolithic and post-Neolithic times, and R-M17 (∼14%), which probably marked several arrivals, at different times, from eastern Eurasia. Hg E, almost exclusively represented by its subclade E-M78, is more common in the Serbs (∼20%) than in Bosniacs (∼13%) and Croats (∼9%), and Hg J, observed in only one Croat, encompasses ∼9% of the Serbs and ∼12% of the Bosniacs, where it shows its highest diversification. By contrast, Hg R-M17 displays similar frequencies in all three groups. On the whole, the three main groups of Bosnia-Herzegovina, in spite of some quantitative differences, share a large fraction of the same ancient gene pool distinctive for the Balkan area.