The Western Interior Seaway (WIS) was a shallow and expansive body of water that covered the central United States during the Late Cretaceous. Attempts to reconstruct temperatures in the seaway using the oxygen isotopic composition of biogenic carbonates have suffered from uncertainty in the oxygen isotopic composition of seawater (δ18Ow) in the semi-restricted basin. We present new reconstructed temperature and δ18Ow data from marine and estuarine environments in the WIS and freshwater environments in WIS source rivers, derived from clumped isotope analyses of bivalve and gastropod shells. We find temperatures of 5–21 °C, δ18Ow values below contemporaneous Gulf of Mexico marine sites, and a strong correlation between δ18Ow and environmental setting. We propose that decreasing δ18Ow values reflect decreasing salinity driven by an increasing contribution of continental runoff. Using a two-end-member salinity-δ18Ow mixing model, we estimate salinities of 29–35 psu (practical salinity units) for the deep marine, 20–32 psu for the shallow marine, and 11–26 psu for the estuarine environments of the WIS. New climate model simulations agree with reconstructed temperatures and salinities and suggest the presence of salinity driven stratification within the seaway.