AbstractBACKGROUNDBlack‐grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post‐emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very‐long‐chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross‐resistance patterns and evolution of this resistance remains poorly understood.RESULTSThe cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black‐grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet‐alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross‐resistance to other VLCFA‐inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA‐inhibitors were not detoxified by the candidate GSTs.CONCLUSIONSAs several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black‐grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross‐resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop‐ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.