AbstractBACKGROUNDRice (Oryza sativa) is an important cereal crop around the world, and has constantly been threaten by the most destructive fungus Magnaporthe oryzae. Pydiflumetofen, a novel succinate dehydrogenase inhibitor (SDHI), is currently being used for controlling various fungal diseases. However, the potential resistance risk of M. oryzae to pydiflumetofen has remained unclear to date, and finding the resistance mechanism is critical for the usage of this fungicide.RESULTSThe M. oryzae strain Guy11 is sensitive to pydiflumetofen, with EC50 value of 1.24 μg mL−1. 58 pydiflumetofen‐resistant (PR) mutants were obtained through pydiflumetofen‐induced spontaneous mutation, with a mean EC50 value >500 μg mL −1, and the resistance factor (RF) >400. The PR mutants displayed positive cross‐resistance to carboxin, but were more sensitive to fluopyram. Sequencing analysis showed that all PR mutants presented a cytosine‐to‐thymine transition at nucleotide position +1218, resulting in a replacement of histidine 245 by tyrosine (H245Y) on MoSdhB. The mutation of MoSdhB exhibited strong resistant phenotype, but no detectable growth deficits in fungal development, including vegetative growth and pathogenicity of M. oryzae. An allele‐specific PCR for rapid detection of the H245Y mutants was established in M. oryzae.CONCLUSIONThe M. oryzae is sensitive to pydiflumetofen, and shows a medium to high resistance risk to pydiflumetofen. A point mutation of MoSdhB (H245Y) is responsible for the fungal resistance to pydiflumetofen in M. oryzae. © 2022 Society of Chemical Industry.