Objectives: Investigation of genetic diversity of the 21 autosomal STR loci included in the GlobalFilerTM PCR Amplification Kit in 529 Pakistani individuals belonging to the Punjabi, Pashtun, Sindhi, Saraiki, and Baloch ethnic groups. Population genetic parameters and forensic informative metrics for each group were evaluated. Results: SE33 showed the greatest power of discrimination in all populations studied. The combined match probability ranged from 8.06E−27 (Saraiki) to 1.05E−26 (Baloch), and the combined power of exclusion ranged from 0.99999999902 (Punjabi) to 0.99999999964 (Pashtun). D12S391 in the Baloch population and D2S441 in the Saraiki population showed deviation from Hardy-Weinberg equilibrium. Conclusion: Significant genetic distances were observed between the Punjabi, Pashtun, and Baloch populations. This study supports the utilization of the GlobalFilerTM STR kit for forensic applications in Pakistan.