Summary. Fetal DNA identification in maternal circulation has provided a new approach for non-invasive prenatal diagnosis. However, fetal DNA can persist in maternal blood long after the delivery, severely hampering this possibility. We addressed the issue of fetal DNA persistence in maternal blood. Thus, we investigated cell-free fetal DNA as a reliable approach in prenatal diagnosis of haemophilia. Forty non-pregnant women, who had had at least a male fetus, 29 control pregnant women, and 14 pregnant women, carriers of hemophilia A or B. The assessment of Y-chromosomal sequences was performed by analysing SRY and amelogenin genes using PCR-based techniques. A protocol consisting of double centrifugation at full speed followed by plasma filtration hampered the detection of Y chromosome-specific sequence in non-pregnant women. In 29 control pregnant women, blinded determination of fetal sex confirmed the specificity and sensitivity of the method applied. In 14 pregnant carriers of hemophilia, the investigation revealed a male fetus in nine pregnancies. Excluding the three cases in which a spontaneous miscarriage occurred, the sensitivity and specificity of fetal sex prediction by SRY and amelogenin gene analyses were both 100% as compared with the invasive approach and the fetal sex outcome at birth (six males and five females). Because of its high accuracy in prediction, fetal gender determination with cell-free fetal DNA in maternal plasma may be a useful tool in prenatal diagnosis of haemophilia allowing for the avoidance of invasive procedures for female fetuses.