The African fig fly (AFF) Zaprionus indianus Gupta, 1970, is an invasive species that has caused significant damage to several monoculture crops since its introduction in the Brazilian territory, resulting losses for farmers and decrease of fruit productions. A way to manage fruit pests is the chemical control, which could act as a selective pressure against genetic variation in the insect populations. The esterases are detoxifying enzymes that can respond to the selection by insecticides, they are conserved in evolution, and are involved in the hydrolysis of esters, including insecticide degradation. Esterases can be identified by isoenzyme electrophoresis, which is a valuable tool for genetic, biochemical, and ecological studies, particularly in approaches with pest species. This experimental study aimed to evaluate survival and esterase polymorphisms at the Est2 and Est3 loci in three different geographical populations of Z. indianus from Brazil (Aracaju, Uberaba and Florianópolis) after exposition to organophosphate malathion. We identified patterns of sex‐specific genetic variation that indicate a response to insecticide exposition. These results indicated that the insecticide biological response in this species is sex‐dependent, providing relevant information about the AFF‐malathion interaction. This study may contribute to the development of more effective and sustainable management strategies for AFF control in Brazil using genetic and ecological approaches.