Glyphosate, currently the world's most extensively used herbicide was on the market for more than 20 years since its introduction in 1975 without reported evolution of resistant weed cases. Glyphosate is the only reported herbicide to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and endogenous accumulation of shikimic acid has been used as a biomarker for the herbicide's activity in plants after spraying. Increased levels of shikimic acid indicate sensitivity or injury while a lack or limited accumulation of shikimic acid shows resistance to glyphosate. Conyza bonariensis seed germinates in flushes in South Africa and its emergence is staggered throughout the year under conducive temperature conditions. This variability in growth stages in the same field poses a challenge in chemical control. Glyphosate herbicide efficacy is affected by environmental conditions, particularly temperature. A glyphosate resistance screening study was carried out based on reports from farmers that C. bonariensis has become increasingly difficult to control especially in conservation tillage systems. This study has confirmed the occurrence of glyphosate-resistant C. bonariensis (hairy fleabane) cases in the western and southern Cape regions of South Africa. A glasshouse screening experiment of 24 biotypes indicates resistance levels ranging from 0.6 to 26.9-fold, with GR50 values of up to 3908.42 g.ae.ha− 1 being reported in a population collected from a vineyard in the Piketberg district of the western Cape. The influence of growth stage and temperature on glyphosate resistance in hairy fleabane was evaluated by using a shikimate assay. Results from this study have shown that response of hairy fleabane to glyphosate is influenced by the phenological stage, with sensitivity or injury decreasing with growth stage. The significant statistical tests verified the dependence of the shikimate pathway on temperature, with more shikimic acid accumulating at 15 °C compared to 27 °C across all biotypes, regardless of origin and glyphosate tolerance status. More tolerance to the herbicide was demonstrated under warm temperature conditions. Possible outcomes of applying glyphosate at the bolting stage and under warm temperature conditions are escapes and no control of susceptible and resistant plants, respectively.