We describe an ancestry-informative autosomal SNP multiplex designed to be a small-scale, flexible panel that can complement uniparental markers in assessing the American variability (i.e. pre-Colombian) found in contemporary indigenous American populations. This study centered on choosing SNPs with the specific characteristics of: 1) extreme allele frequency differences between indigenous Americans and the African, European and East Asian population groups that contribute to present-day population variation in the Americas; 2) high informativeness-for-assignment In values; and 3) well-spaced genomic distribution and chromosomal separation from existing small-scale forensic ancestry marker sets. The resulting capillary electrophoresis SNaPshot single base extension test was named: PIMA (Population Informative Multiplex for the Americas), comprising 26 autosomal SNPs, a single X-chromosome SNP plus the amelogenin sex marker adapted for SNaPshot. PIMA complements the established 34plex forensic ancestry panel to provide a powerful and simple tool for the analysis of American populations, including those with admixed histories, commonly encountered in America. Comparing the results obtained with the combined marker panels of PIMA and 34plex to SNP data from a much larger ancestry panel allowed us to gauge their relative efficiency. PIMA+34plex gives equivalent power to the 314-SNP ?LACE? genomic ancestry control panel, while requiring a much smaller genotyping effort. The ancestry profiles and genetic structure of 22 populations spread across the American continent were estimated using PIMA+34plex data, and those estimates were contrasted with information provided by uniparental markers (mtDNA and Y-chromosome loci) for a small set of admixed individuals from Venezuela. Our results indicate that an American genetic component is efficiently detected in contemporary American populations using a small set of ancestry informative SNPs, and these co-ancestry estimates are consistent with the known history and demography of the Americas. The small scale and high population differentiation power of PIMA, particularly when combined with 34plex, provides a practical and powerful tool for genetic studies of American populations as well as forensic DNA analyses.