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Abstract Zooplankton play an integral role as indicators of water quality in freshwater ecosystems, but exhibit substantial variability in their density and community composition over space and time. This variability in zooplankton community structure may be driven by multiple factors, including taxon-specific migration behavior in response to environmental conditions. Many studies have highlighted substantial variability in zooplankton communities across spatial and temporal scales, but the relative importance of space vs. time in structuring zooplankton community dynamics is less understood. In this study, we quantified spatial (a littoral vs. a pelagic site) and temporal (hours to years) variability in zooplankton community structure in a eutrophic reservoir in southwestern Virginia, USA. We found that zooplankton community structure was more variable among sampling dates over 3 years than among sites or hours of the day, which was associated with differences in water temperature, chlorophyll a, and nutrient concentrations. Additionally, we observed high variability in zooplankton migration behavior, though a slightly greater magnitude of DHM vs. DVM during each sampling date, likely due to changing environmental conditions. Ultimately, our work underscores the need to continually integrate spatial and temporal monitoring to understand patterns of zooplankton community structure and behavior in freshwater ecosystems.