D antigen is the most important and immunogenic antigen of the Rh blood group. The RhD-negative phenotype has different genetic backgrounds with variable distribution in different populations. Hybrid Rhesus box , resulting from RHD gene deletion, is used in genotyping studies of the Rh blood group as a marker to identify the RHD gene deletion. This study for the first time identified genetic mechanisms for the occurrence of RhD-negative phenotype among the Iranian population. 200 RhD-negative blood donors were randomly selected from Tehran Blood Transfusion Center. The phenotype of D, C, Ε, e and c antigens was serologically identified, and DNA was extracted from buffy coat. The molecular analysis of hybrid Rhesus box was performed by PCR-SSP and PCR-RFLP. Moreover, the presence of different exons of RHD gene was investigated by real-time PCR on extracted DNA. Hybrid Rhesus box was detected in all samples, and PCR-RFLP confirmed that 198 (99%) were homozygous for an RHD gene deletion and 2 were heterozygous for hybrid Rhesus box in which one (0.5%) had a weak D type 11 and the other one (0.5%) had a RHD - CE (2 - 9) - D 2 hybrid allele. Similar to Caucasians, the frequency of RHD gene deletion was high among the Iranian population studied in this investigation, so hybrid Rhesus box can be used as an efficient marker to detect RHD gene deletion in our population.