AbstractBACKGROUNDFusarium head blight (FHB) caused by Fusarium graminearum species complex (FGSG) remains a major challenge to cereal crops and resistance to key fungicides by the pathogen threatens control efficacy. Pydiflumetofen, a succinate dehydrogenase inhibitor, and phenamacril, a cyanoacrylate fungicide targeting myosin I, have been applied to combat this disease. Nonetheless, emergence of pydiflumetofen resistance in a subset of field isolates alongside laboratory‐induced facile generation of phenamacril‐resistant isolates signals a critical danger of resistance proliferation.RESULTSOur study investigates the development of dual resistance to these fungicides in F. graminearum. Utilizing pydiflumetofen‐resistant (PyR) and ‐sensitive (PyS) isolates, we obtained dual‐resistant (PyRPhR) and phenamacril‐resistant (PySPhR) mutants on potato sucrose agar containing phenamacril. Mutation rates for phenamacril resistance were comparable between pydiflumetofen‐resistant and ‐sensitive isolates, implying independent pathways for resistance development. The mutants compromised in fungal growth, competitive viability and deoxynivalenol production, suggesting fitness penalties for the dual‐resistant mutants. However, no cross‐resistance was found with tebuconazole or fludioxonil. In addition, we characterized four critical amino acid changes (S217L, C423R, K537T, E420G) in the Myo1 that were verified to confer phenamacril resistance in F. graminearum.CONCLUSIONThis research indicates the possibility of resistance development for both pydiflumetofen and phenamacril in F. graminearum and emphasizes the need for fungicide resistance management for FHB. © 2024 Society of Chemical Industry.