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Background: Determination of foetus rhesus blood group at risk of hemolytic disease has potential application for early non-invasive prenatal testing (NIPT). There are several challenges in developing NIPT rhesus blood group genotyping assays by using cell-free foetal DNA (cff-DNA) in plasma of RhD-negative pregnant women. So, the aim of this study was optimization of Real-time PCR assay for NIPT rhesus genotyping and development of Bi-allelic short insertion/deletion polymorphisms (INDELs) as internal control to optimise and validate rhesus genotyping based on Real-time PCR to avoid false or negative results., Material and Methods: NIPT Rhesus genotyping including RHD (exon 7), RHCc, and RHEe genes were performed by TaqMan Real-time PCR on 104 maternal samples at different gestation ages (12 to ≥40 weeks) from 51 alloimmunized pregnant women. The sensitivity protocol was confirmed with standard DNA samples. Eight selected INDELs were designed and used to detectable cff-DNA in maternal plasma. INDELs frequency and inheritance were determined on 6 family and 61 unrelated individuals. Finally, multiplex Real-time PCR was performed for each sample with INDELs pairs and Rh probes., Results: The results showed 100% accuracy rhesus typing for RHD, RHC and RHE assays and 95.7% accuracy for RHc. Also, eight selected INDELs as internal control for NIPT were 100% concordance for typed samples., Conclusion: The Real-time PCR assay is a suitable method with high sensitivity and specificity for rhesus typing as NIPT for prediction of hemolytic disease in foetuses. The INDELs described here are suitable internal control for confirmation of NIPT on cff-DNA., (© 2022 British Blood Transfusion Society.)