Annual ryegrass (Lolium rigidum) is the only weed species to have evolved resistance to the broad-spectrum herbicide glyphosate in Australia. A population that had failed to be controlled by glyphosate was collected from a vineyard in the Adelaide Hills region of South Australia. Dose–response experiments on this population (SLR 77) showed that it was glyphosate resistant, with an LD50 that was 1.9–3.4 times higher than that of a susceptible population (VLR 1). The movement of radiolabelled glyphosate within SLR 77 plants showed that this population did not have the differential glyphosate translocation mechanism of resistance common to several other Australian glyphosate-resistant populations. Subsequent analysis of shikimic acid accumulation within the plant after glyphosate treatment showed that this population accumulated significantly less shikimic acid than a susceptible population, but more than a glyphosate-resistant population with the translocation mechanism, indicating the possible involvement of another mechanism of resistance. Sequencing of a portion of the SLR 77 5-enolpyruvylshikimate-3-phosphate synthase gene was carried out and a mutation causing an amino acid change at position 106 from proline to threonine was identified. This mutation is likely to be responsible for glyphosate resistance in this population, as mutations in this position have been found to be responsible for glyphosate resistance in goosegrass (Eleusine indica) from Malaysia. This paper represents the first report of target-site glyphosate resistance in L. rigidum and provides evidence that this species has at least two mechanisms of glyphosate resistance present in Australia.