The yellow fever mosquito,Aedes aegypti, particularly in Neotropical regions, is the principal vector of dengue, yellow fever, Zika and Chikungunya viruses. Pyrethroids remain one of the most used insecticides to controlAedesmosquitoes, despite the development of pyrethroid resistance in many mosquito populations worldwide. Here, we report a Brazilian strain ofA. aegyptiwith high levels (approximately 100–60,000 fold) of resistance to both type I and type II pyrethroids. We detected two mutations (V410L and F1534C) in the sodium channel from this resistant strain. This study is the first report of the V410L mutation in mosquitoes. Alone or in combination with the F1534C mutation, the V410L mutation drastically reduced the sensitivity of mosquito sodium channels expressed inXenopusoocytes to both type I and type II pyrethroids. The V410L mutation presents a serious challenge for the control ofA. aegyptiand will compromise the use of pyrethroids for the control ofA. aegyptiin Brazil; therefore, early monitoring of the frequency of the V410L mutation will be a key resistance management strategy to preserve the effectiveness of pyrethroid insecticides.