Resistance to omethoate was suppressible by the hydrolytic enzyme inhibitor SSS-tributyl phosphorotrithioate in a laboratory-selected resistant cotton aphid, Aphis gossypii Glover, strain, suggesting the involvement of hydrolytic enzymes in the detoxification process. The kinetic properties of carboxylesterases from both resistant and susceptible cotton aphids were characterized by four acyl ester substrates: α-naphthyl acetate (α-NA), α-naphthyl butyrate (α-NB), α-naphthyl phosphate (α-NP), and β-naphthyl phosphate (β-NP). No significant differences of carboxylesterase activity were found between resistant and susceptible strains by using either α-NP or β-NP as substrates. In contrast, the susceptible A. gossypii exhibited significantly higher activity compared with resistant aphids with either α-NA or α-NB as substrates. To understand the molecular basis of this esterase-mediated resistance, carboxylesterase genes from both strains were cloned. Two genes share 99.4% identity at the nucleic acid level and 99.2% identity at the amino acid level. The full length of the cDNA opening reading frame is 1581 bp, encoding 526 amino acids. Four amino acid substitutions, Thr²¹⁰→Met²¹⁰, Asn²⁹⁴→Lys²⁹⁴, Gly⁴⁰⁸→Asp⁴⁰⁸, and Ser⁴⁴¹→Phe⁴⁴¹, were identified in the resistant strain. Probing of Southern blots with the 0.5 kb esterase fragment showed the same banding patterns and intensities with genomic DNA extracts from both resistant and susceptible A. gossypii. Furthermore, the MspI and HpaII fragments are the same in both strains, indicating there is no methylation of sequences detected by the probe. The combined results suggest that the structural gene substitution is likely the molecular basis of the organophosphate resistance in this laboratory-selected cotton aphid strain.