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The distributions of mtDNA diversity within and/or among North American haplogroups, language groups, and tribes were used to characterize the process of tribalization that followed the colonization of the New World. Approximately 400 bp from the mtDNA control region of 1 Na-Dene and 33 Amerind individuals representing a wide variety of languages and geographic origins were sequenced. With the inclusion of data from previous studies, 225 native North American (284 bp) sequences representing 85 distinct mtDNA lineages were analyzed. Mean pairwise sequence differences between (and within) tribes and language groups were primarily due to differences in the distribution of three of the four major haplogroups that evolved before settlement of the New World. Pairwise sequence differences within each of these three haplogroups were more similar than previous studies based on restriction enzyme analysis have indicated. The mean of pairwise sequence differences between Amerind members of haplogroup A, the most common of the four haplogroups in North America, was only slightly higher than that for the Eskimo, providing no evidence of separate ancestry, but was about two-thirds higher than that for the Na-Dene. However, analysis of pairwise sequence divergence between only tribal-specific lineages, unweighted for sample size, suggests that random evolutionary processes have reduced sequence diversity within the Na-Dene and that members of all three language groups possess approximately equally diverse mtDNA lineages. Comparisons of diversity within and between specific ethnic groups with the largest sample size were also consistent with this outcome. These data are not consistent with the hypothesis that the New World was settled by more than a single migration. Because lineages tended not to cluster by tribe and because lineage sharing among linguistically unrelated groups was restricted to geographically proximate groups, the tribalization process probably did not occur soon after settlement of the New World, and/or considerable admixture has occurred among daughter populations.