Background: Maternal-fetal Rhd antigen incompatibility causes approximately 50% of clinically significant all oimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal Rhd genotyping in Rhd negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions.Materials and Methods: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal Rhd genotyping, cell free fetal DNA (cffDNA) was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR) for detection of Rhd exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hyper methylated Ras-association domain family member 1 (RASSF1A) gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively.Results: Out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (GA), we correctly diagnosed 45 cases (93.75%) of Rhd positive fetuses and 2 cases (4.16%) of the Rhd negative one. Exon 7 was amplified in one sample, while three other Rhd gene sequences were not detected; the sample was classified as inconclusive, and the Rhd serology result after birth showed that the fetus was Rhd-negative.Conclusion: Our results showed high accuracy of the qPCR method using cffDNA for fetal Rhd genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.