The highly conserved 350-bp major regulatory element HS-40 (or alphaMRE) upstream of the human alpha-globin gene cluster is involved in the regulation of alpha-globin gene expression. The study of alphaMRE differences between human populations and the evolution of alphaMRE sequences in mammals may lead to a better understanding of the function and importance of this element in the regulation of expression of the downstream alpha-cluster. Denaturing gradient gel electrophoresis was used to determine the sequence heterogeneity of the alphaMRE region in 276 unrelated individuals, representing seven different populations. Furthermore, we analysed the alpha major regulatory elements of chimpanzee, orang-utan and rhesus monkeys and compared them with the equivalent human and murine sequences. Six different alphaMRE haplotypes (labelled A to F) were found in humans. Haplotype frequencies between the seven populations showed a gradual shift to a higher haplotype A distribution from west to east, being the highest in Indonesians. The African sample shows the largest divergence in haplotypes. Five out of six different haplotypes were present, three of which were exclusively found in Africans. The high prevalence of the haplotype A in humans, together with the conservation of this haplotype in apes, suggests that it is the ancestral one. The alphaMRE fragment appears to be a highly polymorphic marker, which could be used in combination with the regular markers in the alpha-cluster to extend the haplotype and to follow segregation of alpha-thalassaemia genes in population studies more accurately.