AbstractBackgroundAnnual sowthistle is a weed that is difficult to control in lentil crops in southern Australia due to a lack of herbicide options, widespread herbicide resistance and prolific production of highly mobile seed. This study investigates herbicide resistance in annual sowthistle in the Mid‐North (MN) and Yorke Peninsula (YP) regions of South Australia, identifies and characterizes the mechanisms of acetolactate‐synthase (ALS)‐inhibitor resistance in this amphidiploid species, and combines this with analyses of population structure and gene flow.ResultsALS‐inhibitor‐resistant annual sowthistle is widespread across the YP and MN of South Australia and is associated with a variety of Proline‐197 mutations of the ALS gene, including leucine, alanine, arginine, serine, threonine and histidine. These mutations were found in different combinations on either of the two copies of the ALS gene. An additional 200 tissue samples were collected from across a single field on the YP and the ALS gene was sequenced for all these individuals. Different ALS‐inhibitor resistance profiles were evident between mutation combinations and within mutation combinations, possibly mediated by differing subgenome assortment of the mutations, or altered gene experession of the two ALS homeologs. Population genetics analysis showed evidence of long‐distance dispersal, resulting in highly mobile resistance genes, and multiple instances of resistance mutation evolution.ConclusionsContinuing selection of Sonchus oleraceus populations with ALS‐inhibiting herbicides has resulted in the accumulation of additional mutations within the ALS gene. New practices to control herbicide‐resistant S. oleraceus should be examined, and control should focus on reducing seed set and dispersal to prevent the spread of emerging cases of resistance. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.