BACKGROUND: After the discovery that cell-free fetal DNA (cffDNA) is circulating in the maternal plasma of pregnant women, non-invasive prenatal diagnosis for fetal RhD in maternal plasma in RhD negative women at risk for haemolytic disease of the newborn (HDN) was clinically established and used by many laboratories. The objectives of this study are: (a) to assess the feasibility and report our experiences of the routine implementation of fetal RHD genotyping by analysis of cffDNA extracted from maternal plasma of RhD negative women at risk of HDN, and (b) to estimate the RhD phenotype frequencies, the RHD genotype frequencies and the RhD zygosity in the Cypriot population.METHODS: cffDNA was extracted from maternal plasma of 73 RhD negative pregnant women. Real-Time Multiplex-PCR was used to amplify regions of RHD gene in exons 4, 5 and 10. RhD phenotypes were determined on 445 random samples using conventional agglutination slide test.RESULTS: The fetus was predicted to be positive in 53 cases and negative in 18 cases. Two of cases were identified as D-variants, weak D type-1 and 11. The frequency of RhD negative homozygosity in the Cypriot population was estimated to be 7.2%, while the frequencies of RHD hemizygosity and RhD positive homozygosity was calculated to be 39.2 and 53.6%, respectively.CONCLUSION: Fetal RHD genotyping can be accurately determined using cffDNA from maternal plasma. The implementation of the test has eliminated all use of unnecessary anti-D and reduced the total use of anti-D by 25.3% while achieving appropriate management of the RhD negative pregnancies.