During the late Pleistocene and early Holocene, Bison was widely dispersed across North America and occupied most regions not covered by ice sheets. A dietary study on Bison paleopopulations from Alaska, New Mexico, Florida, and Texas was performed using two methods that relate dental wear patterns to diet, mesowear analysis and microwear analysis. These data were compared to a mixed sample of extant Bison from the North American central plains, extant wood Bison from Alberta (Canada) and a variety of other modern ungulates. Mesowear relates macroscopic molar facet shape to levels of dietary abrasion. The mesowear signature observed on fossil Bison differs significantly from the hyper-abrasive grazing diet of extant Bison. Tooth microwear examines wear on the surface of enamel at a microscopic scale. The microwear signal of fossil samples resembles to modern Bison, but the fossil samples show a greater diversity of features, suggesting that fossil Bison populations regularly consumed food items that are texturally inconsistent with the short-grass diet typical of modern plains Bison. Mesowear and microwear signals of fossil Bison samples most closely resemble a variety of typical mixed feeding ungulates, all with diets that are substantially less abrasive than what is typical for modern plains Bison. Furthermore, statistical tests suggest significant differences between the microwear signatures of the fossil samples, thus revealing geographic variability in Pleistocene Bison diets. This study reveals that fossils are of value in developing an understanding of the dietary breadth and ecological versatility of species that, in recent times, are rare, endangered, and occupy only a small remnant of their former ranges.