During the Holocene, bison (Bison bison) were key components of the Great Plains landscape. This study utilizes serial stable isotope analyses (tooth enamel carbonate) of 29 individuals from five middle Holocene (∽ 7–8.5·ka) archaeological sites to address seasonal variability in movement patterns and grazing behavior of bison populations in the eastern Great Plains. Stable carbon isotopes (δ13C) indicate a bison diet that is similar to the C3/C4 composition of modern tallgrass prairies, while 87Sr/86Sr values generally indicate very little seasonal movement (b50 km) and relatively limited inter-annual movement (b500 km) over the course of 4–5 yr. Analyses of variability in serial stable oxygen isotope samples (δ18O) further substantiate a model of localized bison herds that adhered to upland areas of the eastern Plains and prairie–forest border.