Rigid ryegrass, an important annual weed species in cropping regions of southern Australia, has evolved resistance to 11 major groups of herbicides. Dose–response studies were conducted to determine response of three clethodim-resistant populations and one clethodim-susceptible population of rigid ryegrass to three different frost treatments (−2 C). Clethodim-resistant and -susceptible plants were exposed to frost in a frost chamber from 4:00 P.M. to 8:00 A.M. for three nights before or after clethodim application and were compared with plants not exposed to frost. A reduction in the level of clethodim efficacy was observed in resistant populations when plants were exposed to frost for three nights before or after clethodim application. In the highly resistant populations, the survival percentage and LD50 were higher when plants were exposed to frost before clethodim application compared with frost after clethodim application. However, frost treatment did not influence clethodim efficacy of the susceptible population. Sequencing of the acetyl coenzyme A carboxylase (ACCase) gene of the three resistant populations identified three known mutations at positions 1781, 2041, and 2078. However, most individuals in the highly resistant populations did not contain any known mutation in ACCase, suggesting the resistance mechanism was a nontarget site. The effect of frost on clethodim efficacy in resistant plants may be an outcome of the interaction between frost and the clethodim resistance mechanism(s) present.