Franz Boas organized the collection of information for over 15,000 Native Americans for the World's Columbian Exposition in 1893. Of these individuals, 2482 individuals gave hair samples that are currently stored at the American Museum of Natural History in New York. Now, over 100 years and many technical advances later, the information amassed from these Native American individuals is being reanalyzed to provide a genetic dimension to the analysis of this collection. A new method for the extraction of DNA from aged hair shaft samples was developed and applied in this study. This is the first successful report of research of this kind. Sequence analysis of the mtDNA control region and haplogroup analysis was performed for 19 Mississippi Choctaw and 17 Wisconsin Menominee individuals represented in the Boas hair collection. These samples were then compared with other groups belonging to the same language families for inter-group and intra-group variation. The main objectives of this research were to successfully extract and analyze mitochondrial DNA from these historic hair shafts and to determine if collections such as the Boas are useful for genetic analysis.