The majority of Native Americans nearly exclusively belong to group O of the ABO blood group system. Several hypotheses have been formulated to explain this observation, primarily differing by the presumption that the observed patterns of ABO diversity are due to the processes of the initial peopling of the Americas or due to subsequent events, especially the demographic consequences in the wake of European contact. A promising strategy to reveal possible diachronic ABO frequency changes is the molecular genetic analysis of relevant genetic markers in precontact populations. A previous study by Halverson and Bolnick [Am J Phys Anthropol 137 (2008) 342-347] already accomplished this for indigenous North American populations. Here we present the first study to analyze ABO blood types from pre-Columbian individuals from South America using molecular genetic methods and comparing them to several extant South American, North American, and Siberian populations. We tried to determine ABO blood types for 59 individuals from the southern Peruvian highlands dating to ∼650 to 1250 AD using a newly developed multiplex PCR/SBE assay coamplifying the fragments relevant for blood type determination and three highly discriminating autosomal STRs. Analysis was successful for 31 individuals and revealed that all are exclusively in the O group, predominantly carrying the O02 (01v) allele. No significant difference could be observed between the ancient and modern Native American populations, while all significantly differed from the extant Siberian populations, supporting the suggestion that low ABO diversity results from founder effects during the initial peopling of the Americas. Am J Phys Anthropol 149:242–249, 2012. © 2012 Wiley Periodicals, Inc.