To evaluate the number and time of the migration(s) that colonized the New World we analyzed all available sequences of the first hypervariable segment of the human mitochondrial DNA control region, including 544 Native Americans. Sequence and population trees showed that the Amerind, Na-Dene, and Eskimo are significantly closer among themselves than anyone is to Asian populations, with the exception of the Siberian Chukchi, that in some analyses are closer to Na-Dene and Eskimo. Nucleotide diversity analyses based on haplogroup A sequences suggest that Native Americans and Chukchi originated from a single migration to Beringia, probably from east Central Asia, that occurred approximately 30,000 or approximately 43,000 years ago, depending on which substitution rate is used, with 95% confidence intervals between approximately 22,000 and approximately 55,000 years ago. These results support a model for the peopling of the Americas in which Beringia played a central role, where the population that originated the Native Americans settled and expanded. Some time after the colonization of Beringia they crossed the Alberta ice-free corridor and peopled the rest of the American continent. The collapse of this ice-free corridor during a few thousand years 14,000-20,000 years ago isolated the people south of the ice-sheets, who gave rise to the Amerind, from those still in Beringia; the latter originated the Na-Dene, Eskimo, and probably the Siberian Chukchi.