One of the major insect pyrethroid resistance mechanisms affects its target site, the voltage-gated sodium channel (Na(v)). In Aedes aegypti, the Val1016Ile substitution of the AaNa(v) gene is associated to resistance in several Latin American countries. Genotyping of susceptible and resistant mosquitoes from seven Brazilian localities detected the 1016Ile mutation in five populations with a higher frequency of this substitution in resistant specimens in all cases. Furthermore, analysis of nine additional field populations revealed that five also presented the 1016Ile mutation. Our data suggest a recent dissemination and involvement of this substitution with pyrethroid resistance in Brazil.