Trifloxysulfuron (Envoke) is an acetolactate synthase-inhibitor herbicide and can be used to control many broadleaf weeds and nutsedges in cotton production. However, there is a lack of information on genotypic variation in response to the herbicide. In this field study, 60 Pima (Gossypium barbadense L.) lines, 122 Upland (G. hirsutum L.) lines, and 9 Upland × Pima segregating populations were divided into five tests (18A, 18B, 18G, 18RB, and 18HQ) to evaluate tri-floxysulfuron tolerance at the 7-true leaf stage (42 days after planting) under the same field conditions in 2018. Across the five tests, Pima cotton genotypes tested in this study did not show any visual crop injury based on percentage of plants with chlorosis at 6 days after treatment (DAT ), indicating consistent and high levels of trifloxysulfuron toler-ance. However, the response to trifloxysulfuron within Upland cotton is highly variable. While Upland cotton is overall more sensitive to trifloxysulfuron with crop injury up to 80％ than Pima cotton, 19 lines had injury below 5％ including one line with no visual injury, and 19 lines had injury between 5％ and 10％. In test 18HQ with 15 transgenic Upland cultivars and 17 non-transgenic Upland lines, the analysis of variance detected a significant genotypic difference. The broad-sense heritability estimates for trifloxysulfuron tolerance based on crop injury at 6 DAT was 0.555, suggesting that trifloxysulfuron tolerance in Upland cotton is moderately heritable. This study represents the first report that Pima cotton and many Upland cotton lines are highly tolerant to trifloxysulfuron with no or little crop injury.