Phytoplankton is the essential primary producer in fresh surface water ecosystems. However, excessive phytoplankton growth due to eutrophication significantly threatens ecologic, economic, and public health. Therefore, phytoplankton identification and quantification are essential to understanding the productivity and health of freshwater ecosystems as well as the impacts of phytoplankton overgrowth (such as Cyanobacterial blooms) on public health. Microscopy is the gold standard for phytoplankton assessment but is time-consuming, has low throughput, and requires rich experience in phytoplankton morphology. Quantitative polymerase chain reaction (qPCR) is accurate and straightforward with high throughput. In addition, qPCR does not require expertise in phytoplankton morphology. Therefore, qPCR can be a useful alternative for molecular identification and enumeration of phytoplankton. Nonetheless, a comprehensive study is missing which evaluates and compares the feasibility of using qPCR and microscopy to assess phytoplankton in fresh water. This study 1) compared the performance of qPCR and microscopy in identifying and quantifying phytoplankton and 2) evaluated qPCR as a molecular tool to assess phytoplankton and indicate eutrophication. We assessed phytoplankton using both qPCR and microscopy in twelve large freshwater rivers across the United States from early summer to late fall in 2017, 2018, and 2019. qPCR- and microscope-based phytoplankton abundance had a significant positive linear correlation (adjusted R² = 0.836, p-value < 0.001). Phytoplankton abundance had limited temporal variation within each sampling season and over the three years studied. The sampling sites in the midcontinent rivers had higher phytoplankton abundance than those in the eastern and western rivers. For instance, the concentration (geometric mean) of Bacillariophyta, Cyanobacteria, Chlorophyta, and Dinoflagellates at the sampling sites in the midcontinent rivers was approximately three times that at the sampling sites in the western rivers and approximately 18 times that at the sampling sites in the eastern rivers. Welch's analysis of variance indicates that phytoplankton abundance at the sampling sites in the midcontinent rivers was significantly higher than that at the sampling sites in the eastern rivers (p-value = 0.013) but was comparable to that at the sampling sites in the western rivers (p-value = 0.095). The higher phytoplankton abundance at the sampling sites in the midcontinent rivers was presumably because these rivers were more eutrophic. Indeed, low phytoplankton abundance occurred in oligotrophic or low trophic sites, whereas eutrophic sites had greater phytoplankton abundance. This study demonstrates that qPCR-based phytoplankton abundance can be a useful numerical indicator of the trophic conditions and water quality in freshwater rivers.