Clethodim resistance was identified in 12 rigid ryegrass populations fromwinter cropping regions in four different states of Australia. Clethodim hadfailed to provide effective control of these populations in the field andresistance was suspected. Dose–response experiments confirmed resistance toclethodim and butroxydim in all populations. During 2012, the LD50 of resistant populations ranged from 10.2 to 89.3 g ha−1, making them 3 to 34–fold more resistant to clethodimthan the susceptible population. Similarly, GR50 of resistantpopulation varied from 8 to 37.1 g ha−1, which is 3 to 13.9–foldhigher than the susceptible population. In 2013, clethodim-resistantpopulations were 7.8 to 35.3–fold more resistant to clethodim than thesusceptible population. The higher resistance factor in 2013, especially inmoderately resistant populations, could have been associated with lowerambient temperatures during the winter of 2013. These resistant populationshad also evolved cross-resistance to butroxydim. The resistant populationsrequired 1.3 to 6.6–fold higher butroxydim dose to achieve 50% mortality and3 to 27–fold more butroxydim for 50% biomass reduction compared to thestandard susceptible population. Sequencing of the target-site ACCase geneidentified five known ACCase substitutions (isoleucine-1781-leucine,isoleucine-2041-asparagine, aspartate-2078-glycine, andcysteine-2088-arginine, and glycine-2096-alanine) in these populations. Innine populations, multiple ACCase mutations were present in differentindividuals. Furthermore, two alleles with different mutations were presentin a single plant of rigid ryegrass in two populations.