Aphis gossypii (Glover) has been found to possess multiple mutations in the acetylcholinesterase (AChE) gene (Ace) that might involve target site insensitivity. In vitro functional expression of AChEs reveals that the resistant Ace1 (Ace1R) and Ace2 (Ace2R) were significantly less inhibited by eserine, omethoate, and malaoxon than the susceptible Ace1 (Ace1S) and Ace2 (Ace2S). Furthermore, in both the mutant and susceptible AChEs, Ace2 was significantly less sensitive to eserine, omethoate, and malaoxon than Ace1. These results suggested that both the mutant Ace1 and Ace2 were responsible for omethoate resistance, while the mutant Ace2 played a major role in insecticide resistance. The DNA copy number and transcription level of Ace2 were 1.52- and 1.88-fold higher in the ORR strain than in the OSS strain. Furthermore, the DNA copy number and transcription level of Ace2 were significantly higher than that of Ace1 in either OSS or ORR strains, demonstrating the involvement of Ace2 gene duplication in resistance. Thus, the authors conclude that omethoate resistance in cotton aphids appears to have evolved through a combination of multiple mutations and extensive Ace2R gene duplication. Copyright © 2012 Wiley Periodicals, Inc.