AbstractBACKGROUNDResistance to multiple herbicides is common in Lolium rigidum. Here, resistance to acetolactate synthase (ALS)‐ and susceptibility to acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides was confirmed in a glyphosate‐resistant L. rigidum population (NLR70) from Australia and the mechanisms of pyroxsulam resistance were examined.RESULTSNo ALS target‐site mutations nor gene overexpression were detected. Cytochrome P450 monooxygenase (P450) and glutathione S‐transferase (GST) inhibitors (indicators of some certain P450s or GSTs) did not significantly affect the resistance to pyroxsulam. Nevertheless, HPLC analysis showed that plants of the NLR70 population metabolized pyroxsulam faster than plants of the herbicide‐susceptible population (SVLR1). RNA sequencing analysis and RT‐qPCR validation confirmed that four P450s (CYP709B2, CYP72A14, CYP89A2, CYP94B3), one GT (UGT79), and one ABC transporter (ABCG41) genes were constitutively upregulated in NLR70 plants.CONCLUSIONThis study demonstrates that the glyphosate‐resistant L. rigidum population (NLR70) also exhibits resistance to pyroxsulam and identifies six candidate genes associated with non‐target site resistance to pyroxsulam. © 2025 Society of Chemical Industry.