This study explored the prevalence, genetic diversity, and population structure of azole-resistant Aspergillus fumigatus (ARAf) at Walailak University in Southern Thailand. Three hundred samples were collected from dwellings and workplaces, screened for azole resistance, and tested for drug susceptibility. Molecular detection of alterations in the cyp51A gene and CSP1 typing was performed. Nucleotide polymorphism and haplotype diversity were calculated, and selective neutrality tests were performed. In total, 62 A. fumigatus isolates were identified, with 17 isolates displaying resistance to medical azoles. The prevalence of ARAf in the A. fumigatus isolates was 27.4%. Almost all azole-resistant isolates harboured an amino acid substitution in the hotspot region of the cyp51A gene, especially at or nearby the G54 residue that has been reported as a cause of azole resistance arising from long-term azole treatment. Moreover, some of the ARAf isolates harboured tandem repeats in the promoter region, which have been reported as a cause of resistance arising from the use of azole fungicides in crop protection. Finally, selective neutrality testing also suggested an impact of natural selection on DNA diversity. Therefore, we hypothesize that the factors causing the high prevalence of ARAf in this area are both in vivo- and ex vivo-acquired resistance.