Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response during HCV infections. The goal of this study was to evaluate the association of TLR9 gene polymorphism (rs5743836) in Pakistani patients infected with genotype 3a of HCV. Total 500 subjects were recruited, 400 HCV patients and 100 healthy individuals. Genotyping of TLR9 (-1237T/C, rs5743836) was carried out in 400 HCV patients (323 interferon responders and 77 interferon non responder) and control group by applying High resolution melting (HRM) curve assay. No remarkable differences in distribution of genotype between HCV (p<0.0001; OR= 3.21, 95% CI= (2.514.12) and control groups (p<0.0001; OR=0.092, 95%CI= (0.0580.14) were observed. In conclusion TLR9-1237T/C gene polymorphism may not be considered as a molecular risk for patients with HCV in Pakistan.