Abstract: Analysis of human mitochondrial DNA (mtDNA) is commonly used in ancestry determination due to variation in its Hypervariable (HV) regions. The aim of this study is to amplify HV 1 and 2 regions of human mtDNA to determine individual geographic ancestry using human peripheral blood sample. Twelve pairs of primers were designed using the Primer Premiere 5 Software based on the HV region in human mtDNA. Six human peripheral blood samples (1, 1a, 2, 2a, 3 and 3a) were used in this study. DNA samples were amplified using Polymerase Chain Reaction (PCR) followed by bioinformatics sequence analyses using BLASTn in NCBI. Sequence mutations were analysed using Mitomap to determine the possible haplogroup. Twelve fragments of six DNA samples were successfully amplified and sequenced. Mitomap analysis showed that sample 1 and 1a, 2 and 2a, 3 and 3a come from the same maternal lineage as they share the same sets of mutations in the HV region. Sample 1 and 1a were estimated belongs to haplogroup M10b, sample 2 and 2a belongs to haplogroup F1a and sample 3 and 3a belongs to haplogroup D5b. In conclusion, the twelve primers designed can be used as haplogroup determination hence geographical ancestry.