In Florida, cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is one of the most devastating diseases of cucurbit production. P. cubensis has been shown to overwinter in areas of central and south Florida, which serves as a source of inoculum for cucurbit production in Northern states. Thus, understanding the current population structure of P. cubensis in Florida is vital in properly managing CDM. The objective of the study was to identify differences in seasonal and regional genetic diversity and population structure of P. cubensis, in major cucurbit production areas of Florida. A total of 274 P. cubensis isolates from two cucurbit hosts (cucumber and butternut squash) and three production regions over a two-year period were evaluated. Ten simple sequence repeat (SSR) loci were utilized to establish population genetic statistics and potential structure. High levels of diversity were detected among all isolates based off various diversity estimates. Analysis of molecular variance (AMOVA) analyses revealed significant genetic differences (< 0.001) among host species, production region, and season. STRUCTURE analysis identified two clusters (k=2), which are best explained by cucurbit host species. Results from this study will further our knowledge of genetic variation among P. cubensis populations.