BACKGROUND Mitochondrial DNA (mtDNA) sequencing has been used in many areas, including forensic genetics. Due to the rapid development of sequencing technology, whole mtDNA sequencing is now possible and may be used in epidemiological and forensic studies. This study aimed to use whole mtDNA sequencing to investigate 47 Chongqing Han populations in southwest China and the diversity in the mtGenome reference data. MATERIAL AND METHODS The mtDNA of 47 Chongqing Han populations was generated using the Ion Torrent Personal Genome Machine (PGM) system. The extent of the effects of the mtDNA on the subpopulations was investigated and compared with six other populations from published studies. Pairwise fixation index (FST), a measure of population differentiation due to genetic structure, were calculated. Analysis of molecular variance (AMOVA) was performed, and 1257 hypervariable region data sets were added to the principal component analysis (PCA). RESULTS The whole mtDNA sequencing data of 47 southwest Chinese Han populations were successfully recovered. Expanding the sequencing rage increased the discrimination power of mtDNA from three-times to 25-times based on different populations. The subpopulation effects showed 20 times the differences in match probability when compared with south China regions. CONCLUSIONS Whole mtDNA sequencing distinguished between individuals from 47 Chongqing Han populations in southwest China and has potential applications that include high-quality forensic identification.