Objective：This research was aimed to develop a single-tube multiplex assay of 28 autosomal SNPs to validate its ability of differentiating population from 5 continents and estimate its value in cases. Methods：We selected 28 SNPs from The Global AIMs Nano set to develop the single-tube multiplex assay using SNaPshot technique system. Using this system，we detected 712 individuals from 16 populations in our lab. We combined these results and data that collected from 1 000 genomes and HGDP-CEPH（total 2 804 samples，38 populations）together and used structure cluster analysis and frequency PCA analysis to evaluate this panel. We selected samples of which ancestry component was greater than 90% to build classification library of reference population. Analyses of group matching probability（MP） and individual principal component were performed in 140 individual samples to evaluate the differentiation ability on population source of the multiplex assay in actual samples. Results：Not only could this multiplex assay of 28 SNPs differentiate the population from 5 continents but the admixture of South Asia and Eurasian population. The ancestry component and MP value of some known origin samples were consistent with the sample information. Conclusion：This multiplex assay can differentiate the population from 5 continents and differentiate the ancestry origins of individuals correctly. This multiplex assay is applicable for criminal investigations in the forensic practice.