In this paper, 38 ancestry informative insertion-deletion (InDel) polymorphism markers, with allele frequency differences between three major continental population groups (East Asian, European, and African) were selected from the related literatures and dbSNP database. The Amelogenin locus and a Y chromosomal STR (DYS439) locus were integrated to aid in the sex determination of an unknown sample. All markers were designed and amplified in a single-tube direct amplification assay based on the PCR-CE technology for inferring ancestry of three continental populations. This assay was integrated with a microfluidic system for automatic amplification and InDel genotyping using DNA samples in 1.7 hours. The genotypes of 1607 individuals obtained from the 1000 Genomes Phase III panel were used to assess the differentiation capacity of this 38-plex InDels assay. Then, this assay was validated by sensitivity, genotyping accuracy, and direct PCR ability. 779 samples from 5 populations and 215 direct amplification from saliva and blood samples were genotyped by this assay, then using the structure cluster analysis and principal component analysis to infer ancestral origins and evaluate the accuracy of the system. The results indicate that the 38-plex InDels assay can provide an accurate genotyping, with a sensitivity of 157 pg, and enable the direct amplification of DNA from saliva and blood on filter paper without sample purification. In addition, this assay is sufficient to distinguish between three continental populations and the admixture of Eurasian populations and also, accurately infer the ancestry origins of testing DNA samples. The integration of cell lysis into the microfluidic system is possible to yield ‘sample-in-profile-out’ results of InDel genotyping in 2 hours.