Alu elements, the largest family of interspersed repeats, mobilize throughout the genomes of primates by retroposition. Alu are present in humans in an excess of 500 000 copies per haploid genome. Since some of the insertion alleles have not reached fixation, they remain polymorphic and can be used as biallelic DNA marker systems in investigations of human evolution. In this study, six polymorphic Alu insertional (PAI) loci were used as genetic markers. These markers are thought to be selectively neutral. The presence of these six PAIs was determined by a polymerase chain reaction (PCR)-based assay in 1646 individuals from 47 populations from around the world. Examination of the populations by plotting the first and second principal components, shows the expected segregation of populations according to geographical vicinity and established ethnic affinities. Centroid analysis demonstrated that sub-Sahara populations have experienced higher than average gene flow and/or represent larger populations as compared to groups in other parts of the globe and especially to known inbreed populations. This is consistent with greater heterogeneity and diversity expected of source groups. In addition, maximum likelihood (ML) analyses were performed with these 47 populations and a hypothetical ancestral group lacking the insertion in all six loci. Analysis of our data supports the Out of Africa hypothesis. African populations and admixed groups of African descent formed a single monophyletic group with a basal placement on the tree, which grouped closest to the hypothetical ancestor.