| Summary: | Azole-resistant <i>Aspergillus fumigatus</i> isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the <i>cyp51A</i> gene. Following the discovery of the TR<sub>34</sub>/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the <i>cyp51A</i> gene, indicating that the diversity of resistance mutations in <i>A. fumigatus</i> is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid <i>A. fumigatus</i> to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR<sub>34</sub><sup>3</sup>/L98H evolved from a clinical TR<sub>34</sub>/L98H ancestor isolate, confirmed by whole genome sequencing. TR<sub>34</sub><sup>3</sup>/L98H was associated with increased <i>cyp51A</i> expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR<sub>34</sub><sup>3</sup>, thus supporting the possibility of in-host selection of TR<sub>34</sub> variants.
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