Acetylcholinesterase is an enzyme whose main function is to terminate the transmission of nerve impulses in the cholinergic synapse in insects through the hydrolysis of acetylcholine. However, frequent applications of insecticides can select for individuals with an alteration in the enzyme and functional changes that result in an insensitivity to insecticides. In Mexico, the current strategy to combat insect vectors such as Culex quinquefasciatus is based mainly on the use of synthetic insecticides. The use of these chemicals in health and agricultural practices may also contribute to mosquito insensitivity to several groups of insecticides. Therefore, it is considered necessary to determine if there is any physiological mechanism associated with resistance. For this reason, the objective of this research was to identify if there is insensitivity of acetylcholinesterase to insecticides in Cx. quinquefasciatus populations, collected in urban and rural areas of the State of Yucatán, Mexico. Enzymatic tests were carried out with slight modifications to the established protocols. Areas were identified with mosquito populations showing acetylcholinesterase insensitivity to propoxur, a carbamate insecticide. We consider it important to determine the environmental factors that affect the behavior and response of mosquitoes following exposure to insecticides, and this knowledge could inform the design of efficient strategies in vector control programs.