The mitochondrial DNA (mtDNA) variation in 179 Aleuts from five different islands (Atka, Unalaska, Umnak, St. Paul, and St. George) and Anchorage was analyzed to better understand the origins of Aleuts and their role in the peopling of the Americas. Mitochondrial DNA samples were characterized using polymerase chain reaction amplification, restriction fragment length polymorphism analysis, and direct sequencing of the first hypervariable segment (HVS-I) of the control region. This study showed that Aleut mtDNAs belonged to two of the four haplogroups (A and D) common among Native Americans. Haplogroup D occurred at a very high frequency in Aleuts, and this, along with their unique HVS-I sequences, distinguished them from Eskimos, Athapaskan Indians, and other northern Amerindian populations. While sharing several control region sequences (CIR11, CHU14, CIR60, and CIR61) with other circumarctic populations, Aleuts lacked haplogroup A mtDNAs having the 16265G mutation that are specific to Eskimo populations. R-matrix and median network analyses indicated that Aleuts were closest genetically to Chukotkan (Chukchi and Siberian Eskimos) rather than to Native American or Kamchatkan populations (Koryaks and Itel'men). Dating of the Beringian branch of haplogroup A (16192T) suggested that populations ancestral to the Aleuts, Eskimos, and Athapaskan Indians emerged approximately 13,120 years ago, while Aleut-specific A and D sublineages were dated at 6539 +/- 3511 and 6035 +/- 2885 years, respectively. Our findings support the archaeologically based hypothesis that ancestral Aleuts crossed the Bering Land Bridge or Beringian platform and entered the Aleutian Islands from the east, rather than island hopping from Kamchatka into the western Aleutians. Furthermore, the Aleut migration most likely represents a separate event from those responsible for peopling the remainder of the Americas, meaning that the New World was colonized through multiple migrations.