Fusarium head blight (FHB), caused by Fusarium graminearum, severely affects wheat yield and quality. Fludioxonil, a phenylpyrrole fungicide, is commonly used in the control of FHB. In this study, strains with histidine kinase amino acid substitutions (M402V, M541I, L915M), conferring resistance to fludioxonil, were identified in Hangzhou, Zhejiang Province, China. Through site-directed mutagenesis and structural analysis, we found that point mutations in the HAMP domain (M402V, M541I) cause ultra-high resistance (EC