N-acetyltransferase 2 (NAT2) is responsible for metabolizing xenobiotics; NAT2 polymorphisms lead to three phenotypes: rapid, intermediate and slow acetylators. We aimed to investigate NAT2 diversity in Native Americans. NAT2 exon 2 was sequenced for 286 individuals from 21 populations (Native American and American Mestizos). Excluding the basal/rapid haplotype NAT2∗4, the most frequent haplotypes are NAT2∗5B (35.95%) in hunter-gatherers and NAT2∗7B (20.61%) and NAT2∗5B (19.08%) in agriculturalists that were related to the slow phenotype. A new haplotype was identified in two Amerindians. Data from the ∼44 kb region surrounding NAT2 in 819 individuals from Africa, East-Asia, Europe and America were used in additional analyses. No significant differences in the acetylator NAT2 haplotype and phenotype distributions were found between Native American populations practicing farming and/or herding and those practicing hunting and gathering, probably because of the absence or weakness of selection pressures and presence of demographic and random processes preventing detection of any selection signal.