The molecular basis of an acetyl-CoA carboxylase (ACCase) target-based resistant Lolium rigidum population (WLR 96) was studied here. The carboxyl-transferase domain of the plastidic ACCase gene from resistant individuals was amplified by PCR and sequenced. The DNA sequences were aligned and compared with a susceptible population. Six amino acid substitutions were identified in the resistant population. The substitution Ile-2041-Asn, known to confer resistance to ACCase-inhibiting herbicides aryloxyphenoxypropionate (APP) in Alopecurus myosuroides, was identified in most resistant plants but it is always linked with other amino acid substitutions. This was confirmed by a cleaved amplified polymorphism (CAP) marker and an allele-specific PCR. The sole amino acid substitution Ile-2041-Asn was not found in this population. It is likely this mutation evolved later among individuals already possessing the other substitutions. Three haplotypes were identified from the resistant population based on the six amino acid combinations, and two are linked with herbicide resistance in this population. The multiple amino acid substitutions including the Ile-2041-Asn form the molecular basis endowing a high degree of resistance to ACCase-inhibiting herbicides in this L. rigidum population.