The cotton aphid, Aphis gossypii (Glover), is one of the most serious pests in various vegetable crops. In Korea, some field populations of A. gossypii especially in greenhouse showed high resistance against neonicotinoids. The imidaclopridresistant strain (IR) selected from one of the greenhouse strains was found to be about 3,800 folds more resistant to imidacloprid, compared to the susceptible strain (S), as judged by LC50 values. To identify differentially expressed genes in IR, an isogenic strain, reverse susceptible strain (IRS) was generated from IR and comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from both IR and IRS. Also we confirmed protein expression patterns by 2DE and detoxification enzyme over-expression by synergist test. However there was no significant variation among IR, IRS and S. Comparison of the nucleotide sequence of seven nicotinic acetylcholine receptor (nAChR) subunit (alpha 1-5,7 and beta 1) genes from S and IR strain revealed a point mutation causing an arginine to threonine substitution (R81T) in the loop D region of the nAChR beta 1 subunit of the IR. These mechanisms were also reported in M. persicae and this amino acid change confers a vertebrate-like character to the insect nAChR and results in reduced sensitivity to neonicotinoids. Moreover an extra point mutation, L80S (leucine to serine substitution) was also detected nearby R81T mutation in nAChR beta 1 subunit variant. These mutations can be an additive factor in imidacloprid resistance in A. gossypii. This is the first report of imidacloprid resistance mechanism in A.gossypii. Further, this would be helpful in managing A. gossypii resistant populations in field.