This paper summarizes the current archaeological, physiographic, demographic, molecular, and bioarchaeological understanding of the initial peopling and subsequent population dynamics of South America. Well-dated sites point to a colonization by relatively few broad-spectrum foragers from northeastern Asia between ~13,000 and 12,000 cal BC via the Panamanian Peninsula. By ~11,500–11,000 cal BC, a number of regional, specialized bifacial technologies were developed, with evidence for the seasonal scheduling of resources and the colonization of extreme environments. Restricted mobility, landscape modification, and the cultivation of domesticates were underway by ~8000 cal BC. The early migration routes followed by colonists resulted in a broad east-west population structure among ancient South Americans. Genetic, demographic, and skeletal morphological data indicate that a subsequent demographically driven dispersal into South America largely replaced preexisting central Andeans ~5000 BC, due to increased fertility associated with the shift to agriculture. Beyond the Andes, however, there is little evidence of impact of these later expansions on foragers and horticulturists of the Amazon and Southern Cone who were largely descended from Paleoindians and early Holocene populations.