AbstractCotton (Gossypium hirsutum L.) cultivars resistant to different herbicides have generated important advances in crop management. There is a wide range of cotton resistance to herbicides, however, commercial imidazolinone (IMI)‐tolerant cultivars are not available. In this research, a new mutation that confers tolerance to IMI in cotton was isolated in the M2 generation after seed mutagenic treatments with sodium azide water solutions. The mode of inheritance of the trait was evaluated in the F2 generation by the application of imazethapyr. The inheritance corresponds to a semidominant type with a 1:2:1 distribution of tolerant/intermediate tolerance/not tolerant, in agreement with that observed in most IMI‐tolerant crops. The IMI‐tolerant line behavior was evaluated in the field nursery after the application of the herbicide in the form of foliar and pre‐emergence sprays. The tolerance level was high in pre‐emergence applications and intermediate in foliar applications compared with the nontolerant genotype, which showed a decrease in fiber yield and quality with both methods of application. After sequencing the acetohydroxyacid synthase (AHAS) gene, a point mutation corresponding to Ala205Val in the AHAS enzyme was found, which suggests it could be responsible for the increased IMI tolerance observed in cotton line SP 4172‐32. Based on results from field assays, it can be concluded that this mutant is a promising experimental line that can be used in obtaining IMI‐ tolerant commercial cotton cultivars.