<p>Understanding drivers of phytoplankton assemblage structure is essential given the ecological, aesthetic, and health consequences that vary among taxonomic groups. In this study, we examined phytoplankton assemblage structure over a three-year period in a natural riverine lake in the Upper Mississippi River. Phytoplankton samples along with a suite of limnological variables were collected along a longitudinal gradient of the lake to investigate potential relationships, mechanisms, and drivers between environmental conditions and phytoplankton assemblage structure. We found over 100 phytoplankton taxa and assemblage structure varied between years, months, and sites. With the use of a distance-based linear model, 32.6% of the total variation in phytoplankton assemblages was explained by a combination of chemical, hydrological, and physical variables. Additionally, we noted a one-year absence of the common diatom, <i>Stephanodiscus hantzschii</i>, that may have been related to an unusual spring of warm water temperature and low soluble reactive phosphorus concentrations, indicating possible nutrient limitation in a large river system. Insights into the relationships between environmental conditions and individual and co-occurring species of phytoplankton should aid in developing a greater predictability of assemblages and may enable large river scientists and managers to better anticipate and address water quality conditions under changing climate and hydrologic regimes.</p>