Aspergillus fumigatus is a potentially lethal opportunistic pathogen that causes ~600,000 deaths per year globally. In order to better prevent and treat A. fumigatus infections, it is critical to identify the genomic loci involved in the medically relevant traits. Previously, the molecular mechanisms underlying these traits are mostly investigated using hypothesis-driven gene candidate approach. However, although this approach has yield significant results, it is inherently biased and easily overlooks the genes that are seemingly irrelevant. Here, we used unbiased method, genome-wide association (GWA) analysis, to identify loci involved in azole (a class of antifungal drugs) sensitivity and thermotolerance by analyzing the association between over 100K SNPs and these two traits. We independently conducted GWA for these two traits and discovered a one-gene locus (Afu2g02220) associated with azole sensitivity and a three-gene locus (Afu3g15170, Afu3g15190 and Afu3g15200) associated with growth rate at 52°C, respectively. To functionally validate these two loci, we knocked them out using a CRISPR/Cas9 approach. The Afu2g02220 KO strains grow significantly slower than their parent strain with the presence of azole. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus.