BACKGROUND Widespread reports of reduced efficacy of imidacloprid for managing cotton aphids (Aphis gossypii Glover) in cotton (Gossypium hirsutum L.) prompted an investigation aimed to characterize susceptibility of 43 populations over a 2-year period. The susceptibility of A. gossypii populations to imidacloprid was examined by calculating LC50s. Further analyses related resistance assay results to a gradient of cotton production intensity. RESULTS Concentration-mortality bioassays documented resistance ratios of field collected populations were 4.26 – 607.16 times more resistant than the susceptible laboratory population. There was a significant positive relationship between LC50s and percentage of cotton within 2.5 and 5 km buffers surrounding collection sites. No significant relationship was detected between LC50s and the percentage of alternative crop and non-crop hosts. CONCLUSION Variable and high levels of resistance were detected in A. gossypii populations, and this variation was positively associated with cotton production intensity. Cotton is a host that may receive multiple applications of neonicotinoids (via seed treatment and foliar sprays) annually for seedling and mid-season pests. Rotating modes of action and limiting insecticide use should be implemented to delay the evolution of insecticide resistance in A. gossypii populations. This article is protected by copyright. All rights reserved.