Acetolactate Synthase- (ALS) inhibiting herbicides are important components for the control of ryegrass species infesting cereal-cropping systems worldwide. Although resistance to ALS herbicides in ryegrasses has evolved more than 25 yr ago, few studies have been dedicated to elucidate the molecular mechanisms involved. To this end, we have investigated the molecular basis of chlorsulfuron, sulfometuron-methyl, and imazapyr resistance in AUS5 and AUS23, two ryegrass populations from Australia. Comparison between whole-plant herbicide assays and DNA sequencing results showed that resistance to the nonmetabolizable herbicide sulfometuron-methyl was associated with four different proline mutations at ALS codon position 197 (P197) in AUS23. In addition to three P197 amino acid changes impacting on the efficacies of the two sulfonylurea herbicides, the tryptophan to leucine target-site mutation at ALS codon position 574 (W574L) was present in AUS5, conferring resistance to both sulfometuron-methyl and imazapyr. The samples were also characterized by non target-site-based resistance impacting on the metabolizable herbicide chlorsulfuron only. Interestingly, compound mutant heterozygotes threonine/serine at ALS position 197, and plants with double mutations at positions 197 and 574 were detected, thus reflecting the ability of this outcrossing species to accumulate mutant alleles. Whole-plant dose-response assays conducted on predetermined wild-type and mutant genotypes originating from the same populations allowed for a more precise estimation of the dominant and very high levels of resistance associated with the proline to serine target-site mutation at ALS codon position 197 (P197S) and W574L mutations. The two highly efficient polymerase chain reaction- (PCR) based derived cleaved amplified polymorphic sequence (dCAPS) markers developed here will allow for quick confirmation of 197 and 574 ALS target-site resistance in ryegrass species field samples and also contribute to identify populations characterized by other likely resistance mechanisms in this important weed species.