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Colletotrichum gloeosporioides is the causal pathogen for the devastating walnuts anthracnose. A novel quinone inside inhibitor (QiI) fungicide florylpicoxamid has strong inhibitory efficacy against C. gloeosporioides. This study looked into the resistance risk and mechanism of C. gloeosporioides to florylpicoxamid. The basal level sensitivity of C. gloeosporioides isolates (n = 102) to florylpicoxamid was established with an average 50% mycelial growth inhibition concentration (EC 50 ) value of 0.069 ± 0.035 μg/mL. Six stable florylpicoxamid-resistant mutants with resistance factors of >1000 were produced. The fitness of every mutant was much lower than that of their parental isolates. In general, the resistance risk of C. gloeosporioides to florylpicoxamid would be moderate. Molecular docking results revealed that the amino acid substitutions A37V, and S207L in CgCytb lead to a reduction in the binding affinity between florylpicoxamid and CgCytb, indicating that these two mutations (S207L and A37V in CgCytb) indeed confer florylpicoxamid resistance in C. gloeosporioides. These findings offer a fresh viewpoint on the mechanism underlying QiI fungicide resistance and could support the prudent application of florylpicoxamid in the future to combat walnut anthracnose., (Copyright © 2024 Elsevier Inc. All rights reserved.)