A common waterhemp biotype that was not controlled by triazine or acetolactate synthase (ALS)-inhibiting herbicides was isolated from a field in Bond County, IL, in the fall of 1996. Greenhouse and laboratory experiments determined resistance to atrazine and three ALS-inhibiting herbicides in this biotype. Based on whole-plant response, the Bond County common waterhemp biotype required over 1,000 times more imazethapyr relative to a susceptible biotype to reduce growth 50%. Cross-resistance to thifensulfuron, a sulfonylurea, and flumetsulam, a triazolopyrimidine sulfonanilide, was also detected. Based on in vivo enzyme assays, ALS in the Bond County common waterhemp biotype was 20-, > 8-, and 68-fold less sensitive than ALS in the susceptible biotype to imazethapyr, thifensulfuron, and flumetsulam, respectively. Whole-plant efficacy trials also indicated that the Bond County common waterhemp biotype required more than 20 kg ha−1 of atrazine to inhibit growth 50%. Chlorophyll fluorescence assays revealed that 100 nM atrazine inhibited photosynthesis in the susceptible biotype, whereas 10 M did not affect photosynthesis in the resistant biotype. Regions of the genes encoding ALS and D1 proteins were sequenced to determine the molecular basis for the resistances. Triazine resistance was conferred by a glycine for serine substitution at residue 264 of the D1 protein, while ALS resistance was conferred by a leucine for tryptophan substitution at residue 569 of ALS.