Pseudoperonospora cubensis causes cucurbit downy mildew (CDM), the most economically devastating disease in the Cucurbitaceae. Frequent fungicide applications are required to control the disease in cucumber since the pathogen overcame previously effective host resistance. In this study, we aimed to determine changes in the genetic structure of P. cubensis populations following single-product fungicide applications. Ten previously described microsatellites were used to genotype 880 P. cubensis isolates sampled immediately following spray events during field experiments in 2016 and 2017. Previously described P. cubensis clade and fungicide resistance markers for Carboxylic Acid Amides and Quinone Outside Inhibitors were also used to genotype isolates. Bayesian clustering analysis revealed several genetic clusters in the population. When genetic cluster occurrence was evaluated by treatment, similarities in structure for tolerant varieties with similar genetic background were observed. Likewise, genetic structure was also similar between some fungicides even though treatments belonged to different modes of action. Our results demonstrate the role fungicide applications have in pathogen populations and give insight into the evolution of fungicide resistance in oomycetes. Our findings will ultimately allow for population-informed fungicide recommendations for sustainable disease management of CDM in the United States.