Background: Chemical mosquito control using malathion has been applied in Brazil since 1985. To obtain chemical control efectiveness, vector susceptibility insecticide monitoring is required. This study aimed to describe bioassay standardizations and determine the susceptibility profle of Ae. aegypti populations to malathion and pyriproxyfen, used on a national scale in Brazil between 2017 and 2018, and discuss the observed impacts in arbovirus control. Methods: The diagnostic-doses (DD) of pyriproxyfen and malathion were determined as the double of adult emergence inhibition (EI) and lethal doses for 99% of the Rockefeller reference strain, respectively. To monitor natural populations, sampling was performed in 132 Brazilian cities, using egg traps. Colonies were raised in the laboratory for one or two generations (F1 or F2) and submitted to susceptibility tests, where larvae were exposed to the pyriproxyfen DD (0.03 µg/l) and adults, to the malathion DD determined in the present study (20 µg), in addition to the one established by the World Health Organization (WHO) DD (50 µg) in a bottle assay. Dose-response (DR) bioassays with pyriproxyfen were performed on populations that did not achieve 98% EI in the DD assays. Results: Susceptibility alterations to pyriproxyfen were recorded in six (4.5%) Ae. aegypti populations from the states of Bahia and Ceará, with Resistance Ratios (RR95) ranging from 1.51 to 3.58. Concerning malathion, 73 (55.3%) populations distributed throughout the country were resistant when exposed to the local DD 20 µg/bottle. On the other hand, no population was resistant, and only 10 (7.6%) populations in eight states were considered as exhibiting decreased susceptibility (mortality ratios between 90 and 98%) when exposed to the WHO DD (50 µg/bottle). Conclusions: The feasibility of conducting an insecticide resistance monitoring action on a nation-wide scale was confrmed herein, employing standardized and strongly coordinated sampling methods and laboratory bioassays. Brazilian Ae. aegypti populations exhibiting decreased susceptibility to pyriproxyfen were identifed. The local DD for malathion was more sensitive than the WHO DD for early decreased susceptibility detection.