Author ContributionsRB and AP designed and performed the experiments. RB, AP, TG, SP wrote the manuscript.\n\nOne sentence summaryThis study provides novel insight into herbicide resistance conferred by GST-based detoxification to allow proactive intervention to minimize weed resistance evolution.\n\nAbstractThe evolution of herbicide-resistant weed populations in response to synthetic herbicide selective pressure is threatening safe weed control practices achieved by these molecules. In Australia multiple-resistant populations of annual ryegrass (Lolium rigidum) are effectively controlled by soil-applied herbicides which provide adequate weed control.\n\nIn this study we define the mechanistic basis of the experimentally-evolved resistance to the soil-applied herbicide pyroxasulfone in a L. rigidum population. TLC and HPLC-MS provide biochemical confirmation that pyroxasulfone resistance is metabolism-based with identification and quantification of pyroxasulfone metabolites formed via a glutathione conjugation pathway in pyroxasulfone-resistant L. rigidum plants. The observed over-expression of two putative resistance-endowing GST genes is consistent with pyroxasulfone-resistance in parental plants (P6) and positively correlated to pyroxasulfone resistance in F1 pair-cross progenies. Thus, a major detoxification mechanism involves glutathione conjugation to pyroxasulfone and GST over-expression in pyroxasulfone-resistant L. rigidum plants. The definition of the genetic basis of metabolic resistance in weeds can be a first crucial step towards chemical means to reverse resistance and improve long-term weed resistance management.