Surviving rigid ryegrass plants were collected from a cropping field at Pindar, Western Australia (population WALR 50), after inadequate control by glyphosate applied at the normal field rate. Plants were grown to maturity in pots and seeds were collected. Glyphosate dose–response experiments with known susceptible and resistant control populations confirmed the resistant status of the WALR 50 population. The glyphosate rate resulting in 5% mortality (LD50) and GR50 (the glyphosate rate required to reduce mean growth of individuals to 50% of the untreated control) values for this population were 1,069 and 217 g ae ha−1, respectively, corresponding to R:S ratios of 3.4 and 1.9 for mortality and growth. In addition, a novel root growth–based assay of glyphosate resistance was developed and validated, giving a root growth GR50 R:S ratio of 3.4. A resistance profile was established by assessing population-level survival of WALR 50 after applications at recommended rates of a range of herbicides commonly used for rigid ryegrass control in Australia. High levels of resistance to the acetolactate synthase (ALS)–inhibiting sulfonylurea herbicides chlorsulfuron and sulfometuron, moderate resistance to the acetyl coenzyme A carboxylase (ACCase)–inhibiting herbicide diclofop, and low levels of resistance to the imidazilinone herbicide imazethapyr were found. More detailed dose–response experiments confirmed resistance to chlorsulfuron, sulfometuron, and diclofop. In vitro enzyme-inhibition studies demonstrated that ALS resistance in WALR 50 is due to an insensitive target enzyme and that ACCase resistance is due to a nontarget site–based mechanism. WALR 50 is the first glyphosate-resistant weed population with confirmed resistance to ACCase- and ALS-inhibiting herbicides.