The arrival of bison in North America marks one of the most successful large-mammal dispersals from Asia within the last million years, yet the timing and nature of this event remain poorly determined. Here, we used a combined paleontological and paleogenomic approach to provide a robust timeline for the entry and subsequent evolution of bison within North America. We characterized two fossil-rich localities in Canada’s Yukon and identified the oldest well-constrained bison fossil in North America, a 130,000-y-old steppe bison, Bison cf. priscus. We extracted and sequenced mitochondrial genomes from both this bison and from the remains of a recently discovered, ∼120,000-y-old giant long-horned bison, Bison latifrons, from Snowmass, Colorado. We analyzed these and 44 other bison mitogenomes with ages that span the Late Pleistocene, and identified two waves of bison dispersal into North America from Asia, the earliest of which occurred ∼195-135 thousand y ago and preceded the morphological diversification of North American bison, and the second of which occurred during the Late Pleistocene, ∼45-21 thousand y ago. This chronological arc establishes that bison first entered North America during the sea level lowstand accompanying marine isotope stage 6, rejecting earlier records of bison in North America. After their invasion, bison rapidly colonized North America during the last interglaciation, spreading from Alaska through continental North America; they have been continuously resident since then.