Introduction: In Brasilia, pyriproxyfen (PPF; 0.01 mg/L) has been used for the larval control of Aedes aegypti mosquitoes since 2016. Information on the susceptibility of Ae. aegypti to PPF, and the development of resistance in populations from the Federal District of Brazil (FD) is limited. It is essential to monitor the susceptibility of Ae. aegypti to insecticides in order to improve vector control strategies. This study aimed to evaluate the susceptibility of Ae. aegypti populations from five areas of Brasilia to PPF. Methods: We performed dose-response tests to estimate the emergence inhibition and resistance ratio of each field population, including the Rockefeller reference population. We also analyzed egg positivity, and the density and mortality of larvae and pupae. Results: Populations from Vila Planalto (RR 50 =1.7), Regiment Guards Cavalry (RR 50 =2.5), and Sub-secretary of Justice Complex (RR 50 =3.7) presented high susceptibility to PPF, while the RR values of populations from Lago Norte (RR 50 =7.7) and Varjão (RR 50 =5.9) were moderately high, suggesting the emergence of insipient resistance to PPF in Brasilia. At 30 ng/mL, the highest larvae mortality rate was 2.7% for the population from Lago Norte, while that of pupae was 92.1% for Varjão and Vila Planalto. Conclusions: The five populations of Ae. aegypti from the FD are susceptible to PPF and there is a need to monitor the susceptibility of Ae. aegypti in new areas of the FD.