Y-chromosome variation was analyzed in a sample of 1127 males from the Western Mediterranean area by surveying 16 biallelic and 4 multiallelic sites. Some populations from Northeastern Europe and the Middle East were also studied for comparison. All Y-chromosome haplotypes were included in a parsimonious genealogic tree consisting of 17 haplogroups, several of which displayed distinct geographic specificities. One of the haplogroups, HG9.2, has some features that are compatible with a spread into Europe from the Near East during the Neolithic period. However, the current distribution of this haplogroup would suggest that the Neolithic gene pool had a major impact in the eastern and central part of the Mediterranean basin, but very limited consequences in Iberia and Northwestern Europe. Two other haplogroups, HG25.2 and HG2.2, were found to have much more restricted geographic distributions. The first most likely originated in the Berbers within the last few thousand years, and allows the detection of gene flow to Iberia and Southern Europe. The latter haplogroup is common only in Sardinia, which confirms the genetic peculiarity and isolation of the Sardinians. Overall, this study demonstrates that the dissection of Y-chromosome variation into haplogroups with a more restricted geographic distribution can reveal important differences even between populations that live at short distances, and provides new clues to their past interactions.