|In this study, I examine genetic variation among extant and prehistoric Native Americans to investigate the biological prehistory of eastern North America. The demographic history of the region is reconstructed from patterns of mitochondrial DNA (mtDNA) and Y chromosome diversity, and genetic data are used to test hypotheses about eastern North American prehistory that were formulated based on historical linguistics and the archaeological record. This study also evaluates the relationship between language, culture, geography, and genetic diversity to determine what factors have shaped the patterns of genetic diversity. Specifically, chapter 1 examines mtDNA variation among extant Native Americans from southeastern North America and provides the first evidence that a genetic bottleneck associated with the historical population decline significantly affected Native American genetic variation. Chapter 2 presents a detailed survey of extant Y chromosome diversity in eastern North America and shows that geographic and sociocultural factors have affected Y chromosome variation more than language. When these data are compared with mtDNA data from the same populations, they demonstrate that male and female demographic histories have differed due to post-marital residence patterns and recent male-mediated European admixture. This analysis also addresses the question of Iroquoian origins by suggesting an ancestral Iroquoian homeland in southeastern North America. Finally, chapter 3 examines ancient DNA from two prehistoric burial populations in the Midwest to address questions about the communities participating in the Middle Woodland Hopewell phenomenon. Mitochondrial DNA variation at the Pete Klunk Mound Group in Illinois suggests that Illinois Hopewell communities were matrilocal even though their burial practices were not influenced by matrilineal relationships. This analysis provides no support for a hereditary or ascribed status system in Illinois Hopewell communities. Comparisons with mtDNA data from the Hopewell Mound Group in Ohio demonstrate that migration and gene flow did accompany the cultural exchange between Illinois and Ohio Hopewell communities.