The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most important myiasis-causing flies in South America. It is responsible for severe economic losses to livestock producers, mainly because it causes mortality in newborn calves and reductions in the quality of leather and in the production of milk and meat. The economic losses caused by myiasis, along with those caused by other internal and external parasites, are the main factors limiting meat production. In Brazil, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains. This, associated with the fast development of OP resistance in other myiasis-causing flies, shows the importance of investigating resistance in C. hominivorax. Based on the findings of previous studies, the objective of the current work was to isolate and sequence the E3 gene in C. hominivorax. Mutations at the positions (Gly137 and Trp251) responsible for conferring OP resistance in Lucilia cuprina and Musca domestica L. (Muscidae) were identified in C. hominivorax. In addition, the orthologous region in C. hominivorax contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. The characterization of this gene in natural populations of New World screwworm can be an important tool for monitoring resistance to insecticides throughout its current geographic distribution. This will provide information for the selection and implementation of more effective pest management programmes.