The Tlingit from Southeast Alaska belong to the Northwest Coast cultural tradition, which is defined by regionally shared sociocultural practices. A distinctive feature of Tlingit social organization is the matrilineal exogamous marriage system among clans from two opposite moieties: the Raven/Crow and Eagle/Wolf. Clan and moiety membership are determined by matrilineal descent, and previous genetic studies of Northwest Coast populations have shown a relationship between clan membership and genetic variation of matrilines and patrilines. To further understand this association, in this study mitochondrial DNA sequences from the Tlingit (n = 154) were examined. By comparing mitochondrial DNA with moiety membership information, the authors explore the impact of marriage traditions among the Tlingit with their observable genetic variation. At the genetic level, the results support cultural persistence of Tlingit maternal moiety identity despite the negative impacts of European colonization. This study additionally illustrates the relevance of data derived from Tlingit oral traditions to test hypotheses about population history on the Northwest Coast.