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The demographic tendency in industrial countries to delay child bearing coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis are potent drivers for the development of strategies for non-invasive prenatal diagnosis. A breakthrough has been the discovery of differentially methylated cell free fetal DNA in the maternal circulation. We describe novel bisulfite conversion and methylation-sensitive enzyme digestion DNA methylation related approaches, which we used to diagnose Turner syndrome from first trimester samples. We used an X linked marker, EF3, and an autosomal marker, RASSF1A to discriminate between placenta and maternal blood cell DNA using Real Time methylation specific PCR after bisulfite conversion and Real Time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosomes/autosomes ratio in chorionic villus samples (CVS), thus permitting us to correctly diagnose Turner syndrome samples. The identification of this new marker coupled with the strategy here outlined may be instrumental to develop an efficient, non invasive method of diagnosis of sex chromosomes aneuploidies on plasma samples.