Throughout centuries, the geographic location of the island of Crete has been one of the leading factors shaping the composition of its population. Invasions and commercial and cultural ties at various time periods with European, Middle Eastern, and North African civilizations have created a collage of genetic and/or cultural influences from each of these regions within the island. Previous Y-chromosome diversity analyses uncovered pronounced differences in the frequency distribution of haplogroups from a mountain refugium and surrounding lowland populations of eastern Crete. In this study, the current geographic stratification of mtDNA haplotypes in eastern Crete was explored to elucidate potential sources of maternal gene flow. Our work includes a comparative characterization of two lowland collections from the Heraklion and Lasithi Prefectures in eastern Crete, as well as of an isolated mountain population from the Lasithi Plateau, all three previously examined using Y-chromosome markers. In addition to the presence of European mtDNA haplogroups in all three collections, our analyses reveal a significant contribution of Middle Eastern and Central Asian genetic signatures in the island of Crete, and particularly in the two populations from the Lasithi region at the eastern-most portion of the island. Close association between these Cretan groups and the Balkans can also be discerned, which in the case of the Lasithi Plateau corroborates previously uncovered Y-chromosome affiliations with the same geographic region. Am J Phys Anthropol, 2008. © 2008 Wiley-Liss, Inc.