Abstract: The Fusarium head blight (FHB) caused by Fusarium graminearum, is one of the most notorious diseases during wheat production throughout the world. In this paper, four fludioxonil-resistant mutants (the resistance rate ranged from 318.2 to 782.9) generated by exposure to fludioxonil under laboratory conditions were used as the main research materials to uncover the resistant mechanism of F. graminearum to fludioxonil by biological and molecular biology approaches. All of the resistant mutants were found to have reduced pathogenicity to wheat by about 50% compared to sensitive isolates, especially 2XZ-4R, exhibiting a complete loss of pathogenicity on wheat ears. Meanwhile, all of the mutants were highly sensitive to osmotic stress (0.5 mol/L NaCl, 1.0 mol/L MgCl₂, 1.0 mol/L glucose, or 1.0 mol/L mannitol), and the inhibition rates of mycelial growth were reduced by more than 50% compared to sensitive isolates, indicating that its fitness cost associated with fludioxonil resistance. Furthermore, PAL, POD, and PPO activities of the resistant mutants were increased more than two-fold compared to that of the sensitive isolates. Molecular biology analysis found the expression of the proposed fludioxonil target genes (FgOs1 and FgOs5) were significantly reduced (P＜0.05) in the resistant mutants, indicating that FgOs1 and FgOs5 may be involved in the formation of F. graminearum resistant to fludioxonill. In conclusion, this study explored the biological characteristics of F. graminearum resistant to fludioxonil, and provide new insight into the molecular resistant mechanism of F. graminearum to fludioxonil.