Aedes aegypti and Aedes albopictus are responsible for transmitting major human arboviruses such as Dengue, Zika, and Chikungunya, posing a global threat to public health. The lack of etiological treatments and efficient vaccines makes vector control strategies essential for reducing vector population density and interrupting the pathogen transmission cycle. This study evaluated the impact of long-term pyriproxyfen exposure on the genetic structure and diversity of Ae. aegypti and Ae. albopictus mosquito populations. The study was conducted in Manaus, Amazonas, Brazil, where pyriproxyfen dissemination stations have been monitored since 2014 up to the present day. Double digest restriction-site associated DNA sequencing was performed, revealing that despite significant local population reductions by dissemination stations with pyriproxyfen in various locations in Brazil, focal intervention has no significant impact on the population stratification of these vectors in urban scenarios. The genetic structuring level of Ae. aegypti suggests it is more stratified and directly affected by pyriproxyfen intervention, while for Ae. albopictus exhibits a more homogeneous and less structured population. The results suggest that although slight differences are observed among mosquito subpopulations, intervention focused on neighborhoods in a capital city is not efficient in terms of genetic structuring, indicating that larger-scale pyriproxyfen interventions should be considered for more effective urban mosquito control.