Arylamine N-acetyltranferase 2 is the main enzyme responsible for the isoniazid metabolization into hepatotoxic intermediates and the degree of hepatotoxicity severity has been attributed to genetic variability in the NAT2 gene. The main goal of this study was to describe the genetic profile of the NAT2 gene in individuals from two different regions of Brazil: Rio de Janeiro and Goiás States. Therefore, after preparation of DNA samples from 404 individuals, genotyping of the coding region of NAT2 was performed by direct PCR sequencing. Thirteen previously described SNPs were detected in these Brazilian populations, from which seven: 191 G>A; 282 C>T; 341 T>C; 481 C>T; 590 G>A; 803 A>G and 857 G>A are the most frequent in other populations. The presence of so-called ethnic-specific SNPs in our population is in accordance with the Brazilians' multiple ancestry. Upon allele and genotype analysis, the most frequent NAT2 alleles were respectively NAT2*5B (33%), NAT2*6A (26%) and NAT2*4 (20%) being NAT2*5/*5 the more prevalent genotype (31.7%). These results clearly demonstrate the predominance in the studied Brazilian groups of NAT2 alleles associated with slow over the fast and intermediate acetylator genotypes. Additionally, in Rio de Janeiro, a significantly higher frequency of intermediate acetylation status was found when compared to Goiás (42.5% versus 25%) (p=0.05), demonstrating that different regions of a country with a population characterized by a multi-ethnic ancestry may present a large degree of variability in NAT2 allelic frequencies. This finding has implications in the determination of nationwide policies for use of appropriate anti-TB drugs.