The Upper Cretaceous Western Interior Basin of North America provides a unique laboratory for constraining the effects of spatial climate patterns on the macroevolution and spatiotemporal distribution of biological communities across geologic timescales. Previous studies suggested that Western Interior Basin terrestrial ecosystems were divided into distinct southern and northern communities, and that this provincialism was maintained by a putative climate barrier at ∼50°N paleolatitude; however, this climate barrier hypothesis has yet to be tested. We present mean annual temperature (MAT) spatial interpolations for the Western Interior Basin that confirm the presence of a distinct terrestrial climate barrier in the form of a MAT transition zone between 48°N and 58°N paleolatitude during the final 15 m.y. of the Cretaceous. This transition zone was characterized by steep latitudinal temperature gradients and divided the Western Interior Basin into warm southern and cool northern biomes. Similarity analyses of new compilations of fossil pollen and leaf records from the Western Interior Basin suggest that the biogeographical distribution of primary producers in the Western Interior Basin was heavily influenced by the presence of this temperature transition zone, which in turn may have impacted the distribution of the entire trophic system across western North America.