Resistance to acetolactate synthase (ALS)-inhibitor herbicides due to continuous and repeated selection is widespread in many troublesome weed species, including Palmer amaranth, throughout the United States. The objective of this research was to investigate the physiological and molecular basis of resistance to ALS inhibitors in a chlorsulfuron-resistant Palmer amaranth population (KSR). Our results indicate that the KSR population exhibits a high level of resistance to chlorsulfuron compared with two known susceptible populations, MSS and KSS from Mississippi and Kansas, respectively. MSS is highly susceptible to chlorsulfuron, whereas KSS is moderately sensitive. Dose–response analysis revealed that KSR was more than 275-fold more resistant compared with KSS. Nucleotide sequence analysis of the ALS gene from the plants that survived chlorsulfuron treatment revealed the possibility of evolution of both target site–based and non–target site based resistance to ALS inhibitors in the KSR population. The most common mutation (Pro-197-Ser) in the ALS gene associated with resistance to the sulfonylureas in many weed species was found only in 30% of the KSR population. A preliminary malathion study showed that the remaining 70% of resistant plants might have cytochrome P450–mediated non–target site resistance. This is the first report elucidating the mechanism of resistance to ALS inhibitors in Palmer amaranth from Kansas. Presence of both target site– and non–target site based mechanisms of resistance limits the herbicide options to manage Palmer amaranth in cropping systems.