Phytoplankton provide large quantities of organic carbon and biomolecules that support large river ecosystems. However, when certain groups become overabundant (e.g., cyanobacteria), they can pose a risk to human health and river biota. The purpose of this study was to examine the spatial and temporal dynamics of phytoplankton community composition within the upper Mississippi River. More specifically, we analyzed samples from main channel, impounded, and backwater areas of Navigation Pools 8 and 13 to examine lateral variability within each of these pools. We analyzed samples from the main channel of Pool 26 to examine longitudinal variation among Pools 8, 13, and 26. Phytoplankton and water quality samples were collected during the summer months of 2006–2009. The main channels of Pool 8 and Pool 13 were generally dominated by diatoms, although cyanobacteria were (at times) more abundant. The backwaters were generally dominated by cyanobacteria and typified by flagellated species (e.g., cryptomonads and euglenoids). The main channel of Pool 26 was always dominated by diatoms. Discharge influenced phytoplankton community composition. In Pool 26, taxonomic richness tended to increase with increasing discharge. There were no linear correlations between cyanobacteria total or proportional biovolume and nutrient concentrations, indicating that nutrient limitation was not common. Differences in phytoplankton communities were generally associated with physical factors such as discharge, turbidity, and residence time.