We analyzed the genetic profile of 563 individuals from 12 geographically targeted human populations from Europe, Asia and Africa using 27 human-specific polymorphic Alu insertions. Phylogenetic analyses indicated a clear correspondence between genetic profiles and historical patterns of gene flow and genetic drift. Sub-Saharan African populations (Benin, Cameroon, Kenya and Rwanda) formed a visibly differentiated cluster, indicating the role of the Sahara desert as a strong natural barrier to gene flow. Moreover, a higher than expected genetic affinity between populations from Europe, North Africa and Asia was detected, probably reflecting the homogenizing effects of bidirectional migratory processes between Eurasia and North Africa during the Plio-Pleistocene and Neolithic periods or the insensitivity of these markers in discriminating between these groups. The Ami aborigines of Formosa present a distinctive degree of genetic uniqueness from all the other groups, consistent with a pattern of isolation by distance, small population size and, accordingly, substantial genetic drift. We further tested all 27 Alu loci for their potential usefulness as ancestry informative markers (AIMs). On the basis of differences between weighted allelic frequencies (delta-values) and F(ST) values, we propose that 11 of the 27 Alu elements could be useful as part of the current AIM panels to assess phylogenetic relationships.