A kochia biotype from McDonough County, Illinois, was suspected to be resistant to both triazine and acetolactate synthase (ALS)-inhibiting herbicides. We performed greenhouse and laboratory experiments to confirm, quantify, and determine the molecular basis of multiple herbicide resistance in this biotype. Whole-plant phytotoxicity assays confirmed that the biotype was resistant to triazine (atrazine), imidazolinone (imazethapyr), and sulfonylurea (thifensulfuron and chlorsulfuron) herbicides. Relative to a susceptible kochia biotype, resistance to these herbicides ranged from 500- to > 28,000-fold. The kochia biotype from McDonough County also displayed high levels of resistance (2,000- to 9,000-fold) to ALS-inhibiting herbicides in in vivo ALS enzyme assays, indicating that resistance to these herbicides was site-of-action mediated. Results from chlorophyll fluorescence assays indicated that triazine resistance was also site-of-action mediated. Foliar applications of atrazine had little or no effect on photosynthesis in the resistant biotype, even when atrazine concentrations were 108-fold higher than needed to inhibit photosynthesis in the susceptible biotype. A region of the gene encoding the D1 protein of photosystem II and all of the open reading frame of the gene encoding ALS were sequenced and compared between the resistant and susceptible biotypes. Resistance to triazine and ALS-inhibiting herbicides in the kochia biotype from McDonough County was conferred by, respectively, a glycine for serine substitution at residue 264 of the D1 protein and a leucine for tryptophan substitution at residue 570 of ALS.