Blood plasma of pregnant women contains circulating cell-free fetal DNA (ccffDNA), originating from the placenta. The use of this DNA for non-invasive detection of fetal aneuploidies using massively parallel sequencing (MPS)-by-synthesis has been proven previously. Sequence performance may, however, depend on the MPS platform and therefore we have explored the possibility for multiplex MPS-by-ligation, using the Applied Biosystems SOLiD(™) 4 system. DNA isolated from plasma samples from 52 pregnant women, carrying normal or aneuploid fetuses, was sequenced in multiplex runs of 4, 8 or 16 samples simultaneously. The sequence reads were mapped to the human reference genome and quantified according to their genomic location. In case of a fetal aneuploidy, the number of reads of the aberrant chromosome is expected to be higher or lower than in normal reference samples. To statistically determine this, Z-scores per chromosome were calculated as described previously, with thresholds for aneuploidies set at > +3.0 and < -3.0 for chromosomal over- or underrepresentation, respectively. All samples from fetal aneuploidies yielded Z-scores outside the thresholds for the aberrant chromosomes, with no false negative or positive results. Full-blown fetal aneuploidies can thus be reliably detected in maternal plasma using a multiplex MPS-by-ligation approach. Furthermore, the results obtained with a sample from a pregnancy with 45,X in the cytotrophoblastic cell layer and 46,XX in the mesenchymal core cells show that ccffDNA originates from the cytotrophoblastic cell layer. Discrepancies between the genetic constitution of this cell layer and the fetus itself are well known, and therefore, care should be taken when translating results to the fetus itself.