Spatial synchrony in population dynamics is a ubiquitous ecological phenomenon that can result from predator–prey interactions, synchronized environmental variation (Moran effects), or dispersal. Of these, dispersal historically has been the least well studied in natural systems, partly because of the difficulty in quantifying dispersal in situ. We hypothesized that dispersal routes of plankton were based on the major and consistent water current movements in Kentucky Lake, a large reservoir in western Kentucky, USA. Then, using 26‐year time series collected at 16 locations, we used matrix regression techniques to test whether spatial heterogeneity in strengths of hypothesized dispersal predicted spatial patterns of synchrony of phytoplankton and zooplankton, thereby testing for evidence of dispersal as a possible mechanism of synchrony in this system. Nearly all taxa showed significant spatial synchrony that did not decline with increasing linear distance between locations. All taxa also showed substantial geographic structure in synchrony that was not explained by linear distance. Matrix regression revealed that our hypothesized matrix of dispersal pathways, which differed substantially from linear distance, was a significant predictor of spatial variability in synchrony in phytoplankton biomass, and Bosmina longirostris and Daphnia lumholtzi densities. Thus dispersal was a likely mechanism of synchrony for these taxa. Our hypothesized dispersal matrix was a significant predictor of spatial patterns of synchrony for these taxa even after accounting for numerous alternative possible mechanisms, including possible Moran effects through any of ten physical/abiotic constraints. Our findings indicate that statistically comparing hypothesized or measured dispersal pathway information to synchrony data via matrix regressions can provide valuable evidence for the importance of dispersal as a mechanism of spatial synchrony.