In Tunisia, beta-thalassemia is a common hereditary disease with a carrying rate of 2.21%. Up to now, detection of responsible mutations was made by laborious, expensive, and/or time consuming methods. The aim of this study is to develop and validate a specific assay for detection of the two most frequent mutations in Tunisian population, the IVS-I-110 (G -> A)and Cd39 (C -> T) mutations. In this study, we optimize high resolution melting analysis (HRMA) conditions for these mutations, using control DNAs. Then, we evaluate the strength of this methodology by screening a cohort of patients with beta-thalassemia. All examined reference DNA samples were unambiguously distinguished from each other. For the blinded test, the results were completely compatible with direct sequencing, performed after the HRMA. As HRMA represents a highly sensitive and high-throughput gene scanning method, it can provide timely diagnosis at low cost for effective clinical management of beta-thalassemia.