Abstract: Fusarium crown rot (FCR), predominantly caused by Fusarium pseudograminearum, has been listed as a Category II disease in six provinces of China, posing a significant threat to wheat production. The phenylpyrrole fungicide fludioxonil is a key agent for FCR control. Previous studies indicated that resistance to fludioxonil in F. pseudograminearum is primarily associated with altered expression levels of the FpOS1 gene, which encodes a hybrid histidine kinase. However, the roles of mutations in other FpOS genes and the molecular interactions between FpOS proteins and fludioxonil remain elusive. To address these gaps, we generated 16 fludioxonil-resistant mutants with heritable resistance traits by in vitro selection of four sensitive F. pseudograminearum isolates. These mutants exhibited high resistance levels, with resistance factors (RF) ranging from 633.73 to 8617.07. Compared to their parental isolates, the resistant mutants showed significantly reduced mycelial growth rate, sporulation capacity, and pathogenicity. They were also more sensitive to ionic, osmotic, and oxidative stresses and displayed compromised cell wall and membrane integrity. Fludioxonil demonstrated no cross-resistance with tebuconazole or pydiflumetofen; however, it exhibited weak positive cross-resistance to pyraclostrobin and moderate positive cross-resistance to iprodione. Fludioxonil treatment significantly promoted glycerol synthesis and inhibited deoxynivalenol (DON) production in parental isolates, whereas these regulatory effects were markedly attenuated in the resistant mutants. Mutation analysis identified mutation sites in FpOS1, FpOS4, and FpOS5 genes, with a lower mutation frequency in FpOS1 and no mutations detected in FpOS2. Molecular docking indicated that amino acid substitutions in FpOS4 and FpOS5 significantly reduced the binding affinity of fludioxonil to these target proteins. In conclusion, F. pseudograminearum poses a moderate risk of resistance to fludioxonil. Point mutations in FpOS4 and FpOS5 genes emerge as key molecular drivers of resistance, likely by diminishing the binding affinity between the fungicide and its proteins. This study clarifies the molecular basis of fludioxonil resistance in F. pseudograminearum and provides a scientific rationale for the judicious use of this fungicide in managing FCR.