Mayweed chamomile seeds were collected from six different fields across the Pacific Northwest. All populations (each collection site was considered a population) were suspected to have some level of acetolactate synthase (ALS) resistance. Greenhouse and laboratory studies were conducted to determine if these populations were resistant to three different classes of ALS inhibitors: sulfonylureas (SU), imidazolinones (IMI), and triazolopyrimidines (TP). A whole-plant dose–response and in vitro ALS activity studies confirmed cross-resistance to thifensulfuron + tribenuron/chlorsulfuron (SU), imazethapyr (IMI), and cloransulam (TP); however, resistance varied by herbicide class and population. Two ALS isoforms of the ALS gene (ALS1 and ALS2) were identified in mayweed chamomile; however, only mutations in ALS1 were responsible for resistance. No mutations were found in ALS2. Sequence analysis of the partial ALS gene identified four point mutations at position 197 (Pro197 to Leu, Gln, Thr, or Ser) in the resistant populations. This study demonstrates genotypic variation associated with cross-resistance to ALS inhibitors within and between populations.