Run-of-river dams are often considered to have lower environmental impacts than storage dams due to their smaller reservoirs and low potential for flow alteration. However, this has been questioned for projects recently built on large rivers around the world. Two of the world's largest run-of-river dams-Santo Antônio and Jirau-were recently constructed on the Madeira River, a major tributary to the Amazon River in Brazil. Here we evaluate the effects of the creation of the Santo Antônio dam on the water chemistry and thermal structure of the Madeira River mainstem and back-flooded valleys of tributaries within the reservoir inundated area. In contrast to the mainstem river, some back-flooded tributaries periodically developed thermal stratification, which is associated with higher water residence times. Additionally, biochemical oxygen demand, partial pressure of CO2, and organic carbon all increased in the tributary valleys inundated by the reservoir, possibly due to increased input of allochthonous organic matter and its subsequent mineralization upon back-flooding-a common feature of newly flooded impoundments. The mainstem did not show detectable dam-related changes in water chemistry and thermal structure. Although the majority of the reservoir area maintained riverine conditions, the lateral valleys formed upon back-flooding-corresponding to ~30% of the Santo Antônio reservoir area-developed lake-like conditions akin to a typical reservoir of a storage dam.