Fungal pathogens cause 20% of pre- and post-harvest crop loss and kill more than 1.5 million people a year globally. One of the top four most critical opportunistic fungal pathogens of humans is Aspergillus fumigatus causing 250,000 cases of invasive aspergillosis annually worldwide. A. fumigatus is a ubiquitous saprophyte found in soil, plant debris, and compost. Inhalation of infectious spores by immunocompromised individuals leads to germination of spores, growth of hyphae, and infection in the lungs. Azole drugs are one of the top choices used to combat infections caused by Aspergillus spp. Azoles target Cyp51 (ERG11/Cyp51A/Cyp51B/Cyp51C) in the ergosterol biosynthesis pathway. Ergosterol is a fungal specific sterol that aids in cell membrane fluidity and permeability. Binding of azoles to Cyp51 causes toxic C14 sterol intermediates to accumulate and cause cell death. My thesis focuses on Cyp51 evolution and on azole-resistance in A. fumigatus on the east and west coasts of the United States. I used all publicly available Cyp51 protein sequences on NCBI to better understand the relations among all fungal Cyp51 proteins (chapter 2); and to further investigate the presence of azole-resistant A. fumigatus in the United States (chapter 3). Using A. fumigatus Af293 Cyp51A protein sequence as the query sequence in a database search, we found 435 Cyp51 proteins in 295 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). I used RAxML to create a maximum likelihood tree to investigate how Cyp51 proteins relate to each other. I found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Environmental soil samples of A. fumigatus were taken on the east and west coasts. We sequenced 135 isolates and compared them to publicly available U.S. and global sequences using phylogenetics, PCA, and ADMIXTURE. We found A. fumigatus resistant to azoles and to other agricultural fungicides in 8 states on the east and west coasts with Oregon and Georgia making up most of the samples due to biased sampling. Population structure analyses support three populations of A. fumigatus: Clade 1, Clade 2, and Clade 3.