Background: Aim of this study was to establish the method yielding the highest sensitivity routinely used to determine fetal RhD type and gender from maternal cell-free plasma DNA in different periods of gestation. Methods: Plasma DNA concentrations were measured from 46 pregnant women in different gestational periods and tested for RhD using three different PCR methods on exon 7: Thermal Cycler, Taqman method on LightCycler, and melting curve analysis on LightCycler. In addition, fetal gender was determined by PCR. Cell-free plasma DNA was measured in 100 healthy volunteers as a reference group. Results: The mean value of cell-free plasma DNA in the reference group was 10.9 pg/mL mean, (standard deviation (SD): 3.66) in 50 healthy women and 12.7 pg/mL (SD: 8.2) in 50 healthy men. In the first trimester of pregnancy cell-free plasma DNA was 14.9 pg/mL mean, (SD: 4.2), in the second trimester 15.4 pg/mL mean, (SD: 4.96), and the maximum was achieved in the third trimester of pregnancy 15.6 pg/ml mean, (SD: 6.49). TaqMan probes had the same accuracy, when compared with Thermal Cycler technology (46 samples, 6 failures). Using real-time PCR with melting curve analysis 12 of 17 samples were correctly tested. Gender determination was correctly in 41 of 46 samples. Conclusion: RhD determinations with Taq-Man and Thermal Cycler technology are useful methods for fetal RhD prediction. To increase the accuracy of RhD determination it is necessary to test on other exons in addition.