BACKGROUND:Effective vector control measures are essential in a world where many mosquito-borne diseases have no vaccines or drug therapies available. Insecticidal tools remain the mainstay of most vector-borne disease management programmes, although their use for both agricultural and public health purposes has resulted in selection for resistance. Despite this, little is known about the fitness costs associated with specific insecticide-resistant genotypes and their implications for the management of resistance. In Aedes aegypti, the primary vector of dengue, chikungunya, and Zika, the best-characterised resistance mechanisms are single-point mutations that protect the voltage-gated sodium channel from the action of pyrethroids. METHODS:We evaluated the fitness cost of two co-occurring, homozygous mutations (V1016G and S989P) by back-crossing a resistant strain of A. aegypti from Timor-Leste into a fully susceptible strain from Queensland. The creation of the backcross strain allowed us to isolate these kdr mutations in an otherwise susceptible genetic background. RESULTS:In comparison to the susceptible strain, the backcrossed colony exhibited longer larval development times (5 days, P < 0.001), 24% fewer mosquitoes reached the adult stage (P = 0.005), had smaller wing lengths (females, P = 0.019 and males, P = 0.007) and adult female mosquitoes had a shorter average lifespan (6 days, P < 0.0006). CONCLUSIONS:These results suggest specific and significant fitness costs associated with the double homozygous V1016G/S989P genotype in the absence of insecticides. The susceptibility of a population may recover if the fitness costs of resistant genotypes can be emphasised through the use of insecticide rotations and mosaics or the presence of untreated spatial or temporal refuges.