Bison antiquus was one of the largest and most widely distributed megafaunal species during the Late Pleistocene in North America, giving rise to the modern plains bison in the middle Holocene. Despite the importance of the ancient bison, little is known about its feeding ecology. We employed a combination of extended mesowear, and mesowear III to infer the diet preference and habitat use of three Mexican samples of B. antiquus. Two northern samples from the Transmexican Volcanic Belt morphotectonic Province: La Piedad-Santa Ana and La Cinta-Portalitos, as well as one southern sample from the Sierra Madre del Sur morphotectonic province: Viko Vijin. We found that the northern Mexican samples were primarily non-strict grazers, while the southern sample displays a pattern consistent with mixer feeder habits. This suggests variability among the diets of these bison samples, caused by different paleoenvironments. This evidence complements the paleoenvironmental reconstructions in the studied localities; for the northern samples, open prairies composed of patches of woodland or shrubland and for the southern locality a fluvial floodplain with short-lived vegetation. In both scenarios, grasses (Poaceae) were non-dominant. The dietary habits of our samples of ancient bison in Mexico are the southernmost dietary inference for the species in North America and expand our knowledge of the dietary habits of Bison antiquus during the late Pleistocene.