Background: The chemical control of the mosquito Aedes aegypti, the major vector of dengue, is being seriously threatened due to the development of pyrethroid resistance. Substitutions in the 1016 and 1534 sites of the voltage gated sodium channel (AaNaV), commonly known as kdr mutations, confer the mosquito with knockdown resistance. Our aim was to evaluate the allelic composition of natural populations of Brazilian Ae. aegypti at both kdr sites. Methods: The AaNaV IIIS6 region was cloned and sequenced from three Brazilian populations. Additionally, individual mosquitoes from 30 populations throughout the country were genotyped for 1016 and 1534 sites, based in allele-specific PCR. For individual genotypes both sites were considered as a single locus. Results: The 350 bp sequence spanning the IIIS6 region of the AaNaV gene revealed the occurrence of the kdr mutation Phe1534Cys in Brazil. Concerning the individual genotyping, beyond the susceptible wild-type (NaV S), two kdr alleles were identified: substitutions restricted to the 1534 position (NaV R1) or simultaneous substitutions in both 1016 and 1534 sites (NaV R2). A clear regional distribution pattern of these alleles was observed. The NaV R1 kdr allele occurred in all localities, while NaV R2 was more frequent in the Central and Southeastern localities. Locations that were sampled multiple times in the course of a decade revealed an increase in frequency of the kdr mutations, mainly the double mutant allele NaV R2. Recent samples also indicate that NaV R2 is spreading towards the Northern region. Conclusions: We have found that in addition to the previously reported Val1016Ile kdr mutation, the Phe1534Cys mutation also occurs in Brazil. Allelic composition at both sites was important to elucidate the actual distribution of kdr mutations throughout the country. Studies to determine gene flow and the fitness costs of these kdr alleles are underway and will be important to better understand the dynamics of Ae. aegypti pyrethroid resistance.