Abstract BACKGROUND: Thalassemia is an inherited disorder that is also called mediterranean anemia and is described by a lack of hemoglobin synthesis. β-thalassemia is more common in people of certain origins, particularly those from the Mediterranean region and Arabian Peninsula. The NRAMP1 protein in membranes catalyzes the exit of divalent metal ions from phagolysosomes and their entry into the cytoplasm, including iron and manganese. AIMS: To investigate the possible link between beta-thalassemia major (β-TM) illness and the single-nucleotide polymorphism (3′ untranslated region) rs3 of the NRAMP1 gene. MATERIALS AND METHODS: To investigate the relationship between the condition β-TM and the NRAMP1 gene, the study used restriction fragment length polymorphisms-polymerase chain reaction. The enzyme-linked fluorescent assay was used to quantify the ferritin level. A hematology analyzer was used to quantify hemoglobin concentration. A spectrophotometer technique was used to assess the serum iron, serum total iron-binding capacity (TIBC), and serum unsaturated iron-binding capacity. RESULTS: The results showed a difference in hemoglobin and serum iron levels, serum unbound iron-binding capacity, serum TIBC, and ferritin concentration between individuals suffering from β-thalassemia and healthy groups. In NRAMP1 gene polymorphism, a highly significant difference was observed between the distribution of two alleles (TG− 244 bp, and TG+ 211 bp). CONCLUSION: During an investigation of NRAMP1 gene polymorphism, a significant variance was observed between β-thalassemia and control groups. Those who carried the (244 bp −TG) allele were found to have a 3.6 times higher risk of developing complications of β-TM than those who carried the (211 bp −TG+) allele.