Anthracnose, caused by Colletotrichum truncatum, is an economically important disease in soybean plants. Florylpicoxamid, a novel quinone inside inhibitor (QiI) fungicide, exhibits broad spectrum activity against pathogenic ascomycete fungi. However, little is known about the development of resistance risk to florylpicoxamid and the underlying mechanisms in C. truncatum. In the current study, the sensitivities of C. truncatum isolates to florylpicoxamid were characterized, with an average EC50 value of 0.1540 ± 0.1642 μg/mL. Eleven florylpicoxamid-resistant mutants were obtained, which exhibited fitness comparable to that of the parental isolates. Florylpicoxamid treatment had a lesser impact on ATP production and the activity of the cytochrome bc1 complex in the mutants compared to the parental isolates. Overall, C. truncatum has a medium resistance risk to florylpicoxamid. Additionally, cross-resistance was not observed between florylpicoxamid and the other fungicides tested herein. Three types of point mutations in the Cyt b protein, N31S/A37V, A37V, and S207P, were found to confer varying levels of resistance to florylpicoxamid in C. truncatum, as further proved by molecular docking.