BACKGROUND: Glyphosate is routinely used in Australia to control the Arctotheca species Arctotheca calendula (L.) Levyns (referred hereinafter as capeweed). This study identifies the first global case of field-evolved glyphosate-resistant capeweed, collected from the grainbelt of Western Australia. RESULTS: In 2020, a capeweed biotype that was collected from Borden in the southern Western Australian grainbelt was confirmed to be glyphosate-resistant (referred hereinafter as Spence population). When compared to the pooled mortality of six field-collected, glyphosate susceptible capeweed populations (S1, S2, S3, S4, S5 and S6), the Spence population was found > 11-fold more resistant to glyphosate than the pooled results of the susceptible populations (S1–S6) at the lethal dose of 50% (LD50) level. The growth of the Spence population was also less affected, requiring > 13-fold more glyphosate to reduce growth than the pooled susceptible populations at the growth reduction of 50% (GR50) level. Sequencing of the plastidic 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no known single gene mutation imparting glyphosate resistance. This study, however, did not investigate any other known mechanisms that impart glyphosate resistance. When screened at the field-applied rate, this Spence population was also found to survive an inhibitor of acetolactate synthase (ALS) (metosulam) and an inhibitor of phytoene desaturase (diflufenican). CONCLUSIONS: This is the first confirmation of glyphosate resistance evolution in a capeweed population globally. With capeweed resistance already confirmed to photosystem-I inhibiting herbicides (paraquat and diquat), this study emphasizes the importance of using integrated measures that do not depend only on the use of non-selective herbicides for controlling herbicide resistance-prone capeweed populations.