The most common inherited illness, thalassemia, is thought to have a detrimental effect on public health, particularly in endemic areas. Children with beta thalassemia disease have several mutations. Prevention and premarital examination are still the most effective measures to lessen the burden of beta thalassemia. This study primarily aimed to determine the beta thalassemia carriers in relatives of beta thalassemia major children, the role of gene study in the confirmation of beta thalassemia trait diagnosis, and to detect the genetic defect in the relatives of beta thalassemia major children. The cross-sectional study was conducted on 109 healthy children, aged between six months and 18 years, who were the relatives (second and fourth degree) of beta thalassemia major cases. Gene screening, using the amplification refractory mutation system (ARMS) polymerase chain reaction (PCR), covered the most common 22 alleles in the Mediterranean region, and was successful in detecting 61.5% of beta-globin chain mutations of studied participants, in addition to high prevalence (34.8%) of beta thalassemia carriers among the relatives of beta thalassemia children. The beta thalassemia carrier rate was found to be highly prevalent among relatives of beta thalassemia major children. Despite the accuracy of gene screening in the detection of beta thalassemia carriers, the use of the most common 22 alleles can only detect 61.5% of carriers; hence, the value of tested gene study is still limited in the detection of carrier rates.