BackgroundInsertion/deletion (InDel) analysis plays an indispensable role in human identification, population genetics, and biogeographic research. Profiles of individuals in forensic applications worldwide based on a set of autosomal InDel loci (A‐InDels) in human genomes have been widely used over the past few years.MethodsThe new AGCU InDel 50 Kit contains 47 well‐chosen A‐InDels, ensuring high discriminatory power, and the 2 Y chromosome InDel loci (Y‐InDels) are used for sex determination in case of allele dropout at Amelogenin. In this study, five Northern Han populations residing in different geographic areas of China were recruited and genotyped using the assay.ResultsAfter Bonferroni correction, all 47 A‐InDels were in accordance with the lack of significant departures of Hardy–Weinberg equilibrium in all loci and investigated groups. The combined probability of discrimination and the probability of exclusion in the Han population range from 1–3.2240 × 10–19 to 1–1.3030 × 10–19 and 0.9997, respectively. A comprehensive population genetic relationship investigation between Han Chinese and 26 worldwide populations based on allele frequency correlation was carried out. Our results revealed no significant genetic differentiation in Chinese Han groups. Hierarchical clustering, phylogenetic relationship reconstructions, multidimensional scaling, principal component analysis, and structure analysis were performed, and the results indicated that, genetically, Han populations are closely related to East Asians.ConclusionOverall, this novel 47 A‐InDel assay is a valuable tool that could potentially be used for forensic identification and parentage tests.