ABSTRACTAspergillus fumigatusis a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treatA. fumigatusinfections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms. The predominant mechanisms ofA. fumigatustriazole resistance involve mutations affecting the promoter region or coding sequence of the target enzyme of the triazoles, Cyp51A. However, triazole-resistant isolates withoutcyp51A-associated mutations are frequently identified. In this study, we investigate a pan-triazole-resistant clinical isolate, DI15-105, that simultaneously carries the mutationshapEP88Landhmg1F262del, with no mutations incyp51A. Using a Cas9-mediated gene-editing system,hapEP88Landhmg1F262delmutations were reverted in DI15-105. Here, we show that the combination of these mutations accounts for pan-triazole resistance in DI15-105. To our knowledge, DI15-105 is the first clinical isolate reported to simultaneously carry mutations inhapEandhmg1and only the second with thehapEP88Lmutation.IMPORTANCETriazole resistance is an important cause of treatment failure and high mortality rates forA. fumigatushuman infections. Although Cyp51A-associated mutations are frequently identified as the cause ofA. fumigatustriazole resistance, they do not explain the resistance phenotypes for several isolates. In this study, we demonstrate thathapEandhmg1mutations additively contribute to pan-triazole resistance in anA. fumigatusclinical isolate lackingcyp51-associated mutations. Our results exemplify the importance of and the need for a better understanding ofcyp51A-independent triazole resistance mechanisms.