Identifying resistance‐conferring cytochrome P450s in weeds: experimental strategies and practical considerations

Cytochrome P450 monooxygenases (P450s) play a pivotal role in metabolic herbicide resistance, yet demonstrating that a P450 can metabolize a herbicide is not sufficient to establish that it is responsible for resistance in weeds. The rapid expansion of transcriptomic resources and functional assays has accelerated the discovery of herbicide-metabolizing P450s, creating an increasing need for rigorous criteria to evaluate their actual contribution to resistance. This review examines the evidence required to link individual P450s to resistance phenotypes and discusses how different experimental approaches should be interpreted. We evaluate the strengths and limitations of weed characterization, inheritance analysis, analytical chemistry, transcriptomics, genetic mapping, recombinant protein assays, and plant transformation, emphasizing that no single line of evidence provides definitive proof. Instead, confidence is achieved through the convergence of complementary genetic, biochemical and physiological data. We further argue that the future of metabolic resistance research lies not in cataloguing additional herbicide-metabolizing enzymes, but in identifying the P450s that shape resistance evolution and cross-resistance in agricultural populations. Establishing rigorous criteria for causal inference will improve the biological interpretation of candidate genes and provide a stronger foundation for predicting and managing metabolism-based herbicide resistance. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.