AbstractRice blast caused by Magnaporthe oryzae in cultivated rice is a devastating fungal disease. The crop lost to rice blast is enough to feed more than 60 million people every year. At present, chemical control is still the primary measure to control rice blast. Pyraclostrobin is a methoxy acrylate fungicide, which has the characteristics of broad‐spectrum, high efficiency, environmental protection, and friendliness to non‐target organisms. Pyraclostrobin has been a good option for disease control in China. In this study, M. oryzae resistance to pyraclostrobin was evaluated. The pyraclostrobin‐resistant M. oryzae mutants were screened and the primary fitness parameters of resistant and sensitive strains were investigated. A total of nine pyraclostrobin‐resistant mutants were generated, which exhibited a mutation frequency of 3.46 × 10−4. The EC50 values of the mutants ranged from 2.2185 to 4.8946 μg/ml and resistant factors ranging from 467 to 4504. Biological character evaluations showed that all resistant mutants exhibited decreased fitness in mycelial growth rate, sporulation, and pathogenicity. Positive cross‐resistance was observed between pyraclostrobin and azoxystrobin or picoxystrobin, but not between pyraclostrobin and propiconazole or carbendazim. Sequence analysis of the partial fragment in cytb gene showed that there were two different point mutations in the nine resistant mutants. Seven mutants had a point mutation at position 427 (G to A transition), resulting in the conversion of glycine to serine (G143S). Two other mutants had a point mutation at position 428 (G to C transversion), resulting in the conversion of glycine to alanine (G143A). This mutation site is the main mechanism that has been reported to cause high resistance to QoI fungicides. Therefore, the G143A and G143S mutations found in this study could be the main reason for the mutant resistant to pyraclostrobin.