Abstract: To explore the binding modes of succinate dehydrogenase inhibitor (SDHI) fungicides with Botrytis cinerea succinate dehydrogenase (BcSDH) and elucidate the resistance mechanism of BcSDH to SDHI in structural biology, the three-dimensional structure of BcSDH was constructed by homology modeling. In this work, the resistance mechanism of BcSDH to SDHI was analyzed according to the changes of binding affinity and interactions between five SDHI (isofetamid, fluopyram, luxapyroxad, penthiopyrad and boscalid) and wild-type or mutant BcSDH. The mutation types of BcSDH were predicted by conservative analysis. The results indicated that there was a strong binding interaction between the five SDHI and BcSDH, including hydrophobic action, hydrogen bond, halogen bond, π-π stacking and π-cation interaction. Based on these interactions, the acid part of SDHI inserted into the pocket bottom of BcSDH, and the amine part located outside the pocket. With the B-P225F residue mutation, the pocket narrowed. Therefore, the fungicidal acid part could not enter the pocket. With the B-P225L residue mutation, the binding modes of isofetamid, fluopyram and penthiopyrad with the SDH changed and the binding affinity of them reduced. With the B-H272R residue mutation, the pocket bottom narrowed, which led to binding affinity reduction. In addition, homology analysis showed that B-P225 and B-H272 located in the conservative region of BcSDH, and the B-P225F, B-H272R and B-H272L residue mutations might be random mutations. These results suggested that B-P225F and B-H272R residue mutations of BcSDH migh be the main cause of the resistance of B. cinerea to five fungicides, and might also be one of the main causes of cross-resistance to SDHI. The B-P225L residue mutation may reduce the sensibility of B. cinerea to parts of SDHI, rather than the primary cause of cross-resistance to SDHI. Therefore, reasonable and effective resistance monitoring and management strategies should be adopted to delay the development of the resistance of B. cinerea to SDHI in practical production. And the B-P225F, B-H272R and B-P225L residue mutations should also be considered to avoid cross-resistance in the design of novel SDHI molecules.