Azole resistance inAspergillus fumigatus(AFM) is mainly associated with mutations in CYP51A and its promoter region or its homologue CYP51B. We evaluated the in vitro activity of isavuconazole, itraconazole, posaconazole, and voriconazole against 660 AFM collected during 2017–2020. Isolates were tested via CLSI broth microdilution. CLSI epidemiological cutoff values were applied. Non-wildtype (NWT) isolates to azoles were screened for alterations in the CYP51 sequences using whole genome sequencing. Azoles had similar activities against 660 AFM isolates. Overall, AFM displayed WT MIC values to isavuconazole (92.7%), itraconazole (92.9%), posaconazole (97.3%), and voriconazole (96.7%). Only 66 isolates (10.0%) were NWT to 1 or more of the azoles, and 32 harbored one or more alterations in the CYP51 sequences. Of these, 29/32 (90.1%) were NWT to itraconazole, 25/32 (78.1%) were NWT to isavuconazole, 17/32 (53.1%) were NWT to voriconazole, and 11/32 (34.4%) were NWT to posaconazole. The most frequent alteration was CYP51A TR34/L98H, carried by 14 isolates. Four isolates carried the alteration I242V in CYP51A, and G448S; A9T, or G138C was carried by one isolate each. Multiple alterations in CYP51A were detected in five isolates. Alterations in CYP51B were noted in seven isolates. Among 34 NWT isolates without -CYP51 alterations, WT rates to isavuconazole, itraconazole, voriconazole, and posaconazole were 32.4%, 47.1%, 85.3%, and 82.4%, respectively. Ten different CYP51 alterations were detected in 32/66 NWT isolates. Alterations in AFM CYP51 sequences can have variable effects on the in vitro activity of the azoles that are best delineated by testing all triazoles.