A total of 119 unrelated individuals from two of the major ethnic groups in Ecuador were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex using the SNapShot® assay. Of the above, 42 samples originated from Mestizos (an admixed population) and the remaining 77 were from Native Amerindian Kichwas. We obtained full SNP profiles in all individuals and concordance of duplicated analyses. No deviation from Hardy-Weinberg equilibrium (HWE) was observed for any SNP in the Mestizo and Kichwa populations and only one and four pairs of loci, respectively showed significant linkage disequilibrium. A relatively low genetic diversity and global positive FIS value was observed in Kichwas. A statistically significant global FST value was obtained when the two Ecuadorian populations were compared with populations in Spain, Portugal, Argentina, Denmark, Greenland, China, Somalia and Mozambique. All pairwise FST values were statistically significant. A multi-dimensional scaling based on pairwise FST values showed that the Kichwa population differed from all other populations investigated and that the Mestizos had an intermediate position between Kichwas and Europeans. An admixture analysis indicated that the greater contributor to the Mestizo population was the Kichwas (71.2%) compared to the European contribution. The combined mean match probability and mean paternity exclusion probability were 3.3 × 10-17 and 0.998, respectively, for the Mestizo population and 3.3 × 10-14 and 0.993, respectively, for the Kichwa population.