β-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in β-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real-time PCR assay for copy number analysis of β-globin gene cluster. The copy number of target fragments (i.e. β, δ or Gγ-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire β-globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real-time PCR for analysis of copy number variations in β-globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown β-thalassemia deletions in routine diagnosis of β-thalassemia mutations.