Background <p>In situations where nuclear DNA samples are limited or degraded, mitochondrial DNA can be used as an alternative for forensic applications. The hypervariable (HVRIII) region located on the D-loop of mitochondrial DNA has shown promise in forensic identification due to the presence of unique tandem repeats. In this study, we investigate the variation of Poly-C (HVRII) and Poly-CA repeats (HVRIII) in the Koraga population, a primitive tribe located along the southwestern coast of India.</p> Findings <p>We have identified length heteroplasmy in two specific regions (514–524 and 303–309 nucleotide positions) in the mitochondrial DNA of individuals from the Koraga tribe. Our analysis shows that the (CA)4 and C8 repeats are more frequent in the Koraga population. We have also observed that the Poly-C repeat has a higher discriminatory power (0.6499) compared to Poly-CA (0.5806). Based on the PCA results, the Koragas showed greater genetic affinity with East Asians than South Asians, with Kuntu Koragas clustering uniquely with Urali Kurumans and South Indian Muslims, Onti Koragas bridging the Soppu and Kuntu subgroups, highlighting the importance of studying each Koraga clan separately rather than as a uniform group. </p> Conclusions <p>Our findings suggest that the variations in the Poly-C and Poly-CA repeats in the D-loop of mitochondrial DNA can be used to complement current forensic identification methods. Additionally, our findings provide a new opportunity to investigate selection markers in mitochondrial DNA.</p>