AbstractBACKGROUNDControl of prickly lettuce has become increasingly difficult for lentil growers in southern Australia because of widespread resistance to common herbicides, a lack of alternative herbicide options and the prolific production of highly mobile seed. This study aimed to quantify acetolactate synthase (ALS)‐inhibiting herbicide resistance in the Mid North (MN) and Yorke Peninsula (YP) of South Australia, characterize the resistance mutations present and investigate population structure and gene flow in this species.RESULTSResistance was identified in all populations tested, with average survival of 92% to chlorsulfuron and 95% to imazamox + imazapyr. Five different amino acid substitutions were identified at proline 197 of the ALS gene. There was no significant difference in the median lethal dose (LD₅₀) between plants with these five different substitutions when treated with metsulfuron‐methyl; however, the imidazolinone resistance level was higher in plants with a phenylalanine substitution and lower in plants with a serine. Population structure based on 701 single nucleotide polymorphisms and 271 individuals provided evidence for both independent evolution of the same mutation in different populations, as well as frequent short‐ to medium‐distance dispersal accompanied by occasional long‐distance dispersal events. The overall inbreeding coefficient (F_IS) was calculated at 0.5174, indicating an intermediate level of outcrossing despite the cross‐pollination experiment showing only low outcrossing. In the structure analyses, most individuals from YP were assigned to a single cluster, whereas most individuals from MN were assigned 50% to each of two clusters, indicating some genetic differences between these two regions, but also evidence for dispersal between them.CONCLUSIONSUse of imidazolinone herbicides has selected for mutations conferring higher levels of resistance, such as the Pro‐197‐Phe mutation, and resulted in further spread of resistance in this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.