Botanical gardens have extensive spatial databases of their plant specimens; however, the fungi occurring in them are generally unstudied. Botanical gardens, with their great plant diversity, undoubtedly harbor a wide range of symbiotic fungi, including those that are plant-pathogenic. One such group of fungi is powdery mildews (Erysiphaceae). The powdery mildews are among the most prevalent and economically important plant pathogens in the world, with an estimated 906 species in 19 genera. They are known to infect more than 10,000 species of flowering plants and although some species occur across a range of hosts, many are associated with specific plants. Powdery mildews have undergone a long and dynamic coevolution with their host plants, resulting in co-speciation. Botanical gardens provide a living laboratory in which to study these fungi, leading to a wealth of undiscovered fungal diversity. Furthermore, monitoring pathogens in botanical gardens has led to important ecological findings related to the plant sciences and plant protection. Between 2018 and 2022, a collaborative citizen science project was established with 10 botanical gardens in the United States and Mexico. A total of more than 300 powdery mildew specimens were collected on 220 different host taxa. We sequenced the entire internal transcribed spacer (ITS) and large subunit (LSU) rDNA loci and phylogenetically and morphologically analyzed these collections revealing 130 species, of which 31 are likely unknown to science. This research highlights the importance of botanical gardens as a reservoir of fungal diversity. Future research will further elucidate the coevolutionary relationship between powdery mildews and their hosts and extend the current study to evaluate other plant pathogens and fungi in botanical gardens.