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A highly polymorphic multiplex STR system comprised of HumD21S11, HumFGA and the sex typing system amelogenin has been used to investigate allele frequency distributions in two Canadian Caucasian samplings (British Columbia and Alberta), three Canadian Aboriginal populations (Coastal Salishans from British Columbia and Cree/Ojibwa from Ontario and Saskatchewan) and three ethnic groups from Singapore (Chinese, Malays and Indians). Using the automated fluorescence detection approach on an ABD 373A DNA Sequencer, a total of 20 HumD21S11 and 22 HumFGA alleles were distinguished with a nearly-equal representation of two- and four-base variants. Overall similarities in allele distribution patterns were observed for both STR loci across populations but differences were noted with respect to frequencies. Statistical analysis revealed that 1) both HumD21S11 and HumFGA loci conform to Hardy-Weinberg equilibrium in all eight populations surveyed based on five different tests, and 2) both STR loci are in linkage equilibrium. Results from the 2xN contingency table exact tests for population differentiation demonstrated that the Canadian Caucasian samples from two different provinces were not distinguishable from one another at either STR locus and therefore could be combined to form one Caucasian group. Likewise, Chinese and Malays from Singapore did not show significant differences at either STR locus. In contrast, all other populations examined exhibited differences deemed statistically significant. As a complement to our study, allele frequency distributions were compared between 21 worldwide populations for HumD21S11 and 14 populations for HumFGA. Many alleles never previously observed in worldwide populations, were identified in Canadian Aboriginal and Asian sampling from this study. A total of 24 HumD21S11 and 29 HumFGA alleles were distinguished in worldwide groups. Interesting similarities in allele frequency distribution patterns across populations were noted suggesting that the STR polymorphism at these loci predated the geographical dispersal of ancestral human populations. This study further demonstrates the utility of highly informative STR loci such as HumD21S11 and HumFGA in human population evolutionary history as well as forensic medicine.