Background: Despite the physical and chemical effort to control Aedes aegypti, the arboviruses transmission in the south of Mexico remains latent. Trying to improve the methods of entomological surveillance routinely used, whether the estimation of resistance to insecticides used for its control, as well as their enzyme mechanisms, were influenced by the phase in which the mosquitoes were collected through three different collection methods was investigated. Materials and Methods: Mosquito collections from the “5 de Febrero” neighborhood in Tapachula, Mexico were obtained by ovitraps, larvitraps, and a CDC backpack aspirator. Insecticide resistance of F1 females was determined by WHO diagnostic doses and resistance ratios (RR50), furthermore, levels of insecticide metabolism enzymes were determined by biochemical assays. Results: Overall, in mosquitoes collected by ovitraps, larvitraps, and CDC backpack aspirator respectively, the low mortalities obtained with the discriminant dose to Malathion (27.57%, 26.97%, and 26.91%), and to Bendiocarb (50.5%, 45.36%, and 54.97%) suggest resistance. However, LC50 for Malathion (0.922, 0.934, and 0.915) and for Bendiocarb (0.112, 0.109, and 0.107); and the low resistance ratios (RR50) for Malathion (3.34, 3.29, and 3.27) and for Bendiocarb (2.15, 2.1, and 2.06) does not suggest resistance. Although a slight numerical variation is observed between the three LC50 values, the overlap observed between their confidence intervals allows us to assume that there were no differences between the three methods. In general, esterases (determined with three substrates), glutathion S-transferases (GST) and cytochromes P450 were statistically higher than those of the susceptible strain; and the three enzyme levels were statistically different among the three collection methods (P < 0.01), being those collected with CDC backpack aspirator with the highest levels. Conclusion: Although using a CDC backpack aspirator demonstrated being the best collection method determining a specific resistance mechanism (as elevation at the enzyme level) in the mosquito adult phase, any collection method is reliable to determine whether a field mosquito population is resistant or susceptible to an insecticide.