Forensic DNA investigation is considered the pinnacle of various forensic sciences, as it can extend probabilistic predictions beyond the universe of observations, underpinned by a robust theoretical foundation. Forensic DNA investigations typically involves the development of Short Tandem Repeat (STR) DNA profiles from samples and interpretation of the evidentiary weight of these profiles. The SE33 STR DNA locus, renowned for its global forensic identification applications, is the focus of this study. We assess SE33 polymorphism frequencies, utilizing a sample of 494 unrelated Pakhtun individuals’ genomic DNA which underwent amplification using the GlobalFiler PCR (AmpFlSTR) amplification kit. A total of 44 alleles were observed including few least frequent alleles with a 9.1% homozygote genotype rate and a 90.9% heterozygote genotype rate. Statistical calculations revealed a high power of discrimination (PD) of 0.9921, Power of Exclusion (PE) of 0.814 and a low matching probability (PM) of 0.0078. We further broadened the scope by juxtaposing our allele frequencies with published datasets from neighboring populations. This comparative analysis, not only highlights the unique genetic architecture of the Pakhtun population but also underscores their partial genetic relatedness and distinction from other South Asian populations. The genetic affiliations of Pakhtun population with South Asian communities, enriching our understanding of historical migratory patterns and genetic exchange.

The SE33 locus can be confidently applied in Pakhtun and other population of Pakistan and neighboring countries with shared ancestry across regions.

Keywords: Pakhtun population; Forensic STR; SE33 Locus.