The Aleutian Islands were colonized, perhaps several times, from the Alaskan mainland. Earlier work documented transitions in the relative frequencies of mtDNA haplogroups over time, but little is known about potential source populations for prehistoric Aleut migrants. As part of a pilot investigation, we sequenced the mtDNA first hypervariable region (HVRI) in samples from two archaeological sites on the Alaska Peninsula (the Hot Springs site near Port Moller, Alaska; and samples from a cluster of sites in the Brooks River area near Katmai National Park and Preserve) and one site from Prince William Sound (Mink Island). The sequences revealed not only the mtDNA haplogroups typically found in both ancient and modern Aleut populations (A2 and D2) but also haplogroups B2 and D1 in the Brooks River samples and haplogroup D3 in one Mink Islander. These preliminary results suggest greater mtDNA diversity in prehistoric populations than previously observed and facilitate reconstruction of migration scenarios from the peninsula into the Aleutian archipelago in the past.