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Fusarium head blight (FHB) predominately caused by F. graminearum, is an economical devastating disease for grain cereal crops especially on wheat. The phenylpyrrole fungicide fludioxonil exhibits excellent activity against F. graminearum and has been registered to control FHB in China. In this study, 6 fludioxonil-resistant (FludR) isolates of F. graminearum were identified from 2910 isolates collected from wheat cultivated field in Jiang Su, An Hui and Henan province of China in 2020. The sensitivity assay showed that resistance factor (RF) of FludR isolates ranges from 170.73 to >1000. In comparison with fludioxonil-sensitive (FludS) isolates, all of FludR isolates showed fitness defects in terms of mycelial growth, conidiation and virulence. Under fludioxonil treatment condition, the glycerol accumulation was obviously increased in FludS isolates, but was slightly increased in FludR isolates. Four FludR isolates exhibited increased sensitivity to osmotic stresses. Moreover, there is no positive cross-resistance between fludioxonil and other fungicides including phenamacril, carbendazim and tebuconazole. When treated with fludioxonil, the phosphorylation level of Hog1 was significantly decreased in the four FludR isolates, which was in contrast to the observation in the FludS and two FludR isolates where phosphorylation level of Hog1 was increased. Sequencing assay showed that the mutations were identified in different domains in FgOS1, FgOS2 or FgOS4 in FludR isolates. This was first reported that biological and molecular characterizations of field isolates of F. graminearum resistant to fludioxonil. The results can provide scientific directions for controlling FHB using fludioxonil.