Malays consist of multi sub-ethnic group believed to have different ancestral origins based on their migrations centuries ago. The DNA profiling for every individual in Malaysia is not recorded, making Malaysia lacking in genetic data of its own citizens. This research aimed to study the geographic-ancestry origin of two Malay sub-ethnic population; Kelantan-Malay and Jawa-Malay by looking into the variation of TPA-25 insertion in each population. It specifically studied on several areas of Peninsular Malaysia in the region of Kelantan, Selangor and Johor as the representative of main areas with high percentage of Kelantan-Malay and Jawa-Malay populations. All the data were obtained from an application of TPA-PCR method, forensic parameter (F-statistic) and survey questionnaire that polled genetically on their ancestry origin in each sub-ethnic population. The research showed that population with high percentage of heterozygous allele (Tt) of TPA-25 insertion was likely to have high possibility of genetic drift occurrence. Jawa-Malay showed the highest percentage of heterozygous allele (Tt) with approximately 48% of the population. The FIS value of Kelantan-Malay and Jawa-Malay populations were recorded positive with the values of 0.678 and 0.366 respectively. Moreover, the FIT value recorded was 0.535 which suggested that these two populations were deficits of heterozygotes.