ABSTRACTEuropean bison (Bison bonasus) are the largest endemic vertebrates in Europe, and one of the few megafaunal species to have survived the mass megafaunal extinction during the Pleistocene/Holocene transition (12-9 thousand years ago). Untangling their evolutionary history would provide valuable information about the response of European megafauna to periods of rapid environmental change. However, a severe and recent population bottleneck obscures much of the population history that could be inferred from the genomes of modern individuals. While several studies have attempted to analyse ancient European bison populations directly using ancient DNA, their datasets were limited in temporal and geographic range. In this study we present the most comprehensive dataset of ancient European bison mitochondrial genomes to date, with 131 ancient bison samples from across the Eurasian continent covering over 50 thousand years. We reveal patterns of bison distribution and concurrent environmental changes across a broad geographical and temporal range. In particular, population expansions following periods of extensive forest reduction combined with a decrease in anthropogenic pressures suggest that European bison remain preferentially adapted to an open steppe environment through to the present day.