ABSTRACT. Cochliomyia hominivorax larvae are known for their parasitic habit in living vertebrates, causing considerable economic losses to livestock industry. This ectoparasite has been controlled mainly by applying insecticides, but this method usually results in the selection of resistant individuals. The resistance mechanism known as knockdown resistance (kdr) is a generic term for amino acid substitutions in the sodium channel associated with pyrethroid resistance, and substitutions in residue 251 of the carboxylesterase E3 have been associated with organophosphate and pyrethroid hydrolysis. We looked for L1014F kdr and W251S mutations in the sodium channel and E3 genes, respectively, in C. hominivorax populations. Ten populations obtained from its current distribution were investigated using the polymerase chain reaction-restriction fragment length polymorphism technique. No mutant individuals were found for the kdr mutation. However, the W251S mutation was found in all populations investigated. Results from This correlation was not significant at the highest concentration tested (P = 0.221). We suggest that the W251S mutation in the C. hominivorax carboxylesterase E3 is also associated with pyrethroid hydrolysis. This information may contribute to the elaboration of improved management programs for this ectoparasite.