Hemochromatosis is an autosomal recessive iron overload disorder. The TFR2 gene variant rs7385804 has been implicated in hemochromatosis, but its role in the Pakistani population remains unexplored. This study intended to examine association of TFR2 gene variant rs7385804 with hemochromatosis in Pakistani individuals. We employed a case-control study design, recruiting 200 hemochromatosis patients and 200 healthy controls from Pakistani population. Deoxyribonucleic acid was isolated from blood samples using the phenol-chloroform method. Sanger sequencing and Tetra-ARMS PCR were used to identified the TFR2 genes and its variant rs7385804.Our results showed a significant association between TFR2 gene variant rs7385804 and hemochromatosis in Pakistani population (p < 0.001). The rate of the abnormal alleles was (35%) higher than control (15%). Sanger sequencing confirmed the presence of the variant in 70% of patients, while Tetra-ARMS PCR showed 90% concordance with sequencing results. This study demonstrates the utility of Sanger sequencing and Tetra-ARMS PCR in detecting TFR2 gene variant rs7385804 in Pakistani population. Our findings suggest that this variant may serve as a genetic marker for hemochromatosis in this population. More research is required to authorize these outcomes and discover the clinical consequences of this association.