Azole resistance in Aspergillus fumigatus is most frequently conferred by mutations in the cyp51A gene encoding 14α-sterol demethylases. TR34/L98H and TR46/Y121F/T289A are the two most common mutations associated with environmental resistance selection. We studied the minimal inhibitory concentration (MIC) distribution of clinical A. fumigatus isolates to characterize the Clinical and Laboratory Standards Institute (CLSI) susceptibility profiles of isolates with the wild-type (WT) cyp51A genotype, and isolates with the TR34/L98H and TR46/Y121F/T289A cyp51A mutations. Susceptibility testing was performed according to CLSI M38-A2. The MICs of 363 A. fumigatus isolates were used in this study. Based on the CLSI epidemiological cut-off values (ECVs), 141 isolates were phenotypically non-WT and 222 isolates had a phenotypically WT susceptibility. All isolates with the TR34/L98H mutation had an itraconazole MIC > 1 mg/L which is above the CLSI ECV. Eighty-six of 89 (97%) isolates with the TR34/L98H mutation had voriconazole and posaconazole MICs above the CLSI ECV, i.e., MICs of 1 and 0.25 mg/L, respectively. The isolates with a TR46/Y121F/T289A mutation showed a different phenotype. All 37 isolates with a TR46/Y121F/T289A mutation had a voriconazole MIC above the CLSI ECV, while 28/37 (76%) isolates had an itraconazole MIC > 1 mg/L. Interestingly, only 13 of 37 (35%) isolates had a posaconazole MIC > 0.25 mg/L.