AbstractBACKGROUNDFusarium pseudograminearum is one of the dominant pathogens of Fusarium crown rot (FCR) worldwide. Unfortunately, no fungicides have yet been registered for the control of FCR in wheat in China. Pydiflumetofen, a new‐generation succinate dehydrogenase inhibitor, exhibits excellent inhibitory activity to Fusarium spp. A resistance risk assessment of F. pseudograminearum to pydiflumetofen and the resistance mechanism involved have not yet been investigated.RESULTSThe median effective concentration (EC50) value of 103 F. pseudograminearum isolates to pydiflumetofen was 0.0162 μg mL−1, and the sensitivity exhibited a unimodal distribution. Four resistant mutants were generated by fungicide adaption, which possessed similar or impaired fitness compared to corresponding parental isolates based on the results of mycelial growth, conidiation, conidium germination rate, and virulence determination. Pydiflumetofen showed strong positive cross‐resistance with cyclobutrifluram and fluopyram but no cross‐resistance with carbendazim, phenamacril, tebuconazole, fludioxonil, or pyraclostrobin. Sequence alignment revealed that pydiflumetofen‐resistant F. pseudograminearum mutants had two single‐point mutations of A83V or R86K in FpSdhC1. Molecular docking further confirmed that point mutation of A83V or R86K in FpSdhC1 could confer resistance of F. pseudograminearum to pydiflumetofen.CONCLUSIONFusarium pseudograminearum shows an overall moderate risk of developing resistance to pydiflumetofen, and point mutation FpSdhC1A83V or FpSdhC1R86K could confer pydiflumetofen resistance in F. pseudograminearum. This study provided vital data for monitoring the emergence of resistance and developing resistance management strategies for pydiflumetofen. © 2023 Society of Chemical Industry.