Acetyl-coenzyme A carboxylase (ACCase) inhibitors such as haloxyfop, pinoxaden and clethodim selectively control grass weed species in many types of crop. Here, we characterised two populations of Lolium multiflorum (Italian ryegrass), R and RV, from South Canterbury and Central Otago respectively, that were suspected to be resistant to ACCase-inhibiting herbicides. Based on 50% reduction in biomass (GR(50)), in two dose-response experiments, both populations were highly resistant to haloxyfop with resistance factors (RF) >400 times, but resistance to pinoxaden was low (RF of 2). Furthermore, population R was found to be moderately resistant to clethodim (RF >4, based on 50% reduction in survival (LD50)), but population RV had RFs barely greater than 1, based on GR(50). Gene sequencing showed that the same target site mutation (Ile-2041-Asn) was associated with resistance to the three herbicides in both populations. The zygosity of the resistance alleles in the plants tested could cause the differences in resistance to clethodim observed between the populations, even though they carried the same mutation. This is the first report of the mechanism of resistance to ACCase-inhibiting herbicides in Italian ryegrass from New Zealand.