The arrival of Bison in North America significantly affected competitive relationships among herbivores and may have played an important role in the eventual extinction of contemporaneous Pleistocene megafaunal taxa. To investigate the dietary ecology of fossil bison and examine the potential for such an ecological impact, we analyzed the stable carbon and oxygen isotope composition of Bison latifrons and Bison antiquus tooth enamel from four regions with distinct climatic settings and vegetation. Newly sampled regions include American Falls, Idaho; Diamond Valley Lake, California; Central Florida; and Snowmastodon, Colorado. The Snowmastodon site contains the highest elevation assemblage known to contain B. latifrons. We compared this new isotopic dataset to all available published isotopic data for Pleistocene Bison sp. B. latifrons and B. antiquus were found to have isotopically similar diets when recovered from the same regions. Both species of bison consumed C4 vegetation but incorporated significant proportions of C3 plants into their diets based on mean δ13C values. Independent paleobotanical records support the interpretation that the majority of specimens exhibited mixed feeding behavior. We detected statistically significant differences in bison diets between regions that correspond to mean annual temperatures. Serial oxygen sampling indicates different degrees of seasonal variation in the meteoric waters consumed by bison in each of the newly sampled regions. Bison from Diamond Valley Lake and Snowmastodon exhibit the strongest signals of seasonal differences in vegetation consumption, while animals from American Falls and Central Florida consumed similar vegetation year-round. Our results confirm the hypothesis that Pleistocene Bison exhibited more malleable dietary and habitat requirements than previously assumed. This dietary plasticity allowed Pleistocene bison to compete with other species of the North American megafauna until their eventual replacement by the modern Bison bison. •Fossil bison consumed C4 vegetation but incorporated significant proportions of C3 plants into their diets when available.•Pleistocene bison from California and Colorado exhibit strong signals of seasonal variation in vegetation consumption.•Fossil bison from Idaho and Central Florida exhibit isotopic signals indicating consistent diets year-round.•Isotopic data from new localities indicate a wide range of climate conditions and dietary flexibility in Pleistocene bison