Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides affect fatty acid biosynthesis in plants and are widely used to control smooth and hare barley in dicot crops in Australia. Recently, growers have experienced difficulty in controlling smooth and hare barley with herbicides from this mode of action. Dose–response experiments conducted on five suspected resistant populations confirmed varying levels of resistance to quizalofop and haloxyfop. The level of resistance in these populations was greater than 27-fold to quizalofop and greater than 15-fold to haloxyfop. The quizalofop dose required to reduce shoot biomass by 50% (GR50) for the resistant populations varied from 52.6 to 111.9 g ha−1, and for haloxyfop from 26.5 to 71.3 g ha−1. Sequencing the CT domain of the ACCase gene from resistant plants of different populations confirmed the presence of previously known mutations Ile1781Leu and Gly2096Ala. Amino acid substitution at the 2096 position conferred a greater level of resistance to haloxyfop than the substitution at the 1781 position. This study documents the first known case of field-evolved target-site resistance to ACCase-inhibiting herbicides in Australian populations of smooth barley.