A population of common ragweed not controlled by an acetolactate synthase (ALS)-inhibiting herbicide, cloransulam-methyl, was sampled near Dunkirk, IN, the first year of the herbicide's commercialization in 1998. Resistance in the Dunkirk population was confirmed by treating greenhouse-grown seedlings with cloransulam-methyl. ALS activity assays and DNA sequencing were used to identify the resistance mechanism. ALS isolated from plants of the Dunkirk population exhibited an R/S ratio for cloransulam-methyl of >5,000 when compared to ALS activity of populations from Claire City, SD, and V & J Seed Farms. R/S ratios of 4,100 and 110 were observed for two other ALS-inhibiting herbicides, chlorimuron and imazaquin, respectively. DNA sequencing revealed that an inferred leucine for tryptophan substitution at amino acid position 574 in ALS was responsible for the observed herbicide resistance. Additionally, DNA sequencing revealed significant variability among common ragweed ALS alleles. Two fragments of ALS were sequenced from three plants each of the Claire City and Dunkirk populations, totaling 688 nucleotide base pairs, of which 72 were polymorphic.