A population of giant ragweed not controlled by cloransulam was identified near Seymour, IN, during the first year of that herbicide's commercialization in 1998. Results from acetolactate synthase (ALS) activity assays performed by Dow AgroSciences showed that resistance was caused by an altered ALS. Studies were conducted to define more precisely the molecular basis of resistance and to determine cross-resistance to other ALS-inhibiting herbicides. Sixteen greenhouse-grown giant ragweed plants from the Seymour population were tested individually with postemergence (POST) applications of cloransulam, imazethapyr, or chlorimuron, or by using a nondestructive leaf disk assay to determine resistant or sensitive herbicide responses. All plants identified from the Seymour population as resistant to cloransulam were cross-resistant to imazethapyr and chlorimuron. Two DNA fragments, totaling 804 nucleotide base pairs, within ALS were sequenced from each of the 16 plants. Sequence data, combined with phenotypic data, showed that a tryptophan to leucine substitution at amino acid position 574 of ALS (based on numbering of the Arabidopsis ALS) was responsible for ALS-inhibitor resistance. Among 11 resistant and 5 sensitive giant ragweed plants analyzed from the Seymour population, at least 15 different ALS alleles were identified. Of these 15 alleles, two alleles, at an average frequency of 0.25, contained a leucine at position 574 and conferred resistance. The 13 alleles that conferred susceptibility to ALS-inhibiting herbicides occurred at an average frequency of 0.04.