Genetic association studies can be used to identify factors that may contribute to disparities in disease evident across different racial and ethnic populations. However, such studies may not account for potential confounding if study populations are genetically heterogeneous. Racial and ethnic classifications have been used as proxies for genetic relatedness. We investigated genetic admixture and developed a questionnaire to explore variables used in constructing racial identity in two cohorts: 50 African Americans and 40 Nigerians. Genetic ancestry was determined by genotyping 107 ancestry informative markers. Ancestry estimates calculated with maximum likelihood estimation were compared with population stratification detected with principal components analysis. Ancestry was approximately 95% west African, 4% European, and 1% Native American in the Nigerian cohort and 83% west African, 15% European, and 2% Native American in the African American cohort. Therefore, self-identification as African American agreed well with inferred west African ancestry. However, the cohorts differed significantly in mean percentage west African and European ancestries (P < 0.0001) and in the variance for individual ancestry (P ≤ 0.01). Among African Americans, no set of questionnaire items effectively estimated degree of west African ancestry, and self-report of a high degree of African ancestry in a three-generation family tree did not accurately predict degree of African ancestry. Our findings suggest that self-reported race and ancestry can predict ancestral clusters but do not reveal the extent of admixture. Genetic classifications of ancestry may provide a more objective and accurate method of defining homogenous populations for the investigation of specific population-disease associations.