Riverine ecosystems receive organic matter (OM) from terrestrial sources, internally produce new OM, and biogeochemically cycle and modify organic and inorganic carbon. Major gaps remain in the understanding of the relationships between carbon sources and processing in river systems. Here we synthesize isotopic, elemental, and molecular properties of dissolved organic carbon (DOC), particulate organic carbon (POC), and dissolved inorganic carbon (DIC) in the Upper Mississippi River (UMR) system above Wabasha, MN, including the main stem Mississippi River and its four major tributaries (Minnesota, upper Mississippi, St. Croix, and Chippewa Rivers). Our goal was to elucidate how biological processing modifies the chemical and isotopic composition of aquatic carbon pools during transport downstream in a large river system with natural and man‐made impoundments. Relationships between land cover and DOC carbon‐isotope composition, absorbance, and hydrophobic acid content indicate that DOC retains terrestrial carbon source information, while the terrestrial POC signal is largely replaced by autochthonous organic matter, and DIC integrates the influence of in‐stream photosynthesis and respiration of organic matter. The UMR is slightly heterotrophic throughout the year, but pools formed by low‐head navigation dams and natural impoundments promote a shift toward autotrophic conditions, altering aquatic ecosystem dynamics and POC and DIC compositions. Such changes likely occur in all major river systems affected by low‐head dams and need to be incorporated into our understanding of inland water carbon dynamics and processes controlling CO2 emissions from rivers, as new navigation and flood control systems are planned for future river and water resources management. Key Points Dissolved organic carbon composition is linked to basin forest area and wastewater inputs and shows little in‐stream biological modification Biological processes modify dissolved inorganic carbon isotopic composition, so that it deviates from terrestrial carbon source composition Low‐head dams and reservoirs shift fluvial systems toward autotrophic conditions, reflected in particulate organic carbon composition Plain Language Summary Watersheds of the Upper Mississippi River Basin drain a region of diverse land use types, ranging from heavily agricultural to forested. This study investigates links between the chemical features of carbon carried by these rivers and different land use types within their basins. These features are used to identify signals of biological activity during downstream transport, which influence how much carbon dioxide the river releases to the atmosphere. We found that certain portions of the carbon carried by the Upper Mississippi River maintain markers of land use, while other portions reflect the activity of photosynthesis and respiration. River impoundments such as the natural Lake Pepin on the Mississippi River main stem remove light‐blocking particles, thus shifting the river's balance from respiration toward photosynthesis. These results are important for understanding how rivers may respond to future changes in land use, climate, and other environmental conditions, especially given the expected global increase in river navigation structures.