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BACKGROUND Grape anthracnose caused by an ascomycete fungus Colletotrichum gloeosporioides is widely controlled by demethylation-inhibitors (DMIs) for decades in China. The resistance status and mechanism of C. gloeosporioides against DMIs is not well understood. RESULTS All the difenoconazole-resistant (DfnR ) isolates from vineyards exhibited the decreased fitness. Positive cross-resistance was detected between DMI-triazoles. Sequence alignment results from the DfnR and DfnS isolates revealed that multiple mutations are distributed at CgCYP51A, concomitant with mutations at CgCYP51B. The EC50 values of single deleted and complemented mutants of CgCYP51A and CgCYP51B showed that ΔCgCYP51A became more sensitive to difenoconazole, but not ΔCgCYP51B. Furthermore, all the single complemented mutants had a stronger biological fitness than the progenitor strain. All the defectives of ΔCgCYP51A and ΔCgCYP51B could be restored by the complementation of the whole corresponding gene from the resistant strains. The relative gene expressions of CgCYP51A and CgCYP51B in most of the mutants were greatly upregulated relative to the progenitor isolate when treated with difenoconazole at the same concentration. Moreover, the extension of 5-AAs (GNETI) caused by the mutation at stop codon of CgCYP51A, concurrent with other seven amino acid substitutions and the synonymous mutation P10P (CCG → CCT), significantly enhanced the DMI-resistance. CONCLUSION The DMI-resistance of C. gloeosporioides selected in vineyards is conferred by mutations at CgCYP51s, and validated by genetics method. Roles of CgCYP51A and CgCYP51B are overlapped, and reversely balanced, but cannot be replaced reciprocally. This article is protected by copyright. All rights reserved.