A number of redroot pigweed and Powell amaranth populations from various locations in Ontario, Canada, have distinct patterns of resistance to the acetolactate synthase–inhibiting herbicides imazethapyr and thifensulfuron. This suggested the presence of diverse ALS gene mutations among these populations. Seven polymerase chain reaction primer pairs were used to amplify the gene to obtain full sequence information and to determine the identity of resistance-conferring mutations. There was a high degree of similarity in the ALS gene of the two species with only five nucleotides and one amino acid differing. A total of four herbicide resistance-conferring mutations were identified in the two species. The Ala122Thr, Ala205Val, and Trp574Leu amino acid substitutions were found in redroot pigweed whereas Ala122Thr, Trp574Leu, and Ser653Thr were detected in Powell amaranth. The pattern of resistance known to be conferred by the mutations concurred with the resistance level observed at the whole plant level. Distinct mutations being found in geographically separated populations suggest that selection for resistance occurred simultaneously in different locations. It reinforces the fact that resistance to ALS inhibitors is easily selected and that growers need to take this into account when formulating weed management strategies.