To develop informative tools for the study of population affinities in African Americans, we sequenced the hypervariable segments I and II (HVS I and HVS II) of mitochondrial DNA (mtDNA) from 96 Sierra Leoneans; European Americans; rural, Gullah-speaking African Americans; urban African Americans living in Charleston, South Carolina; and Jamaicans. We identified single nucleotide polymorphisms (SNPs) exhibiting ethnic affinities, and developed restriction endonuclease tools to screen these SNPs. Here we show that three HVS restriction site polymorphisms (RSPs), EcoRV, FokI, and MfeI, exhibit appreciable differences in frequency (average delta = 0.4165) between putative African American parental populations (i.e., extant Africans living in Sierra Leone and European Americans). Estimates of European American mtDNA admixture, calculated from haplotypes composed of these three novel RSPs, show a cline of increasing admixture from Gullah-speaking African American (m = 0.0300) to urban Charleston African American (m = 0.0689) to West Coast African American (m = 0.1769) populations. This haplotype admixture in the Gullahs is the lowest recorded to date among African Americans, consistent with previous studies using autosomal markers. These RSPs may become valuable new tools in the study of ancestral affinities and admixture dynamics of African Americans.