Purpose:To prospectively validate a quantitative fluorescent polymerase chain reaction (PCR) assay as a method of rapid prenatal aneuploidy detection for chromosomes 13, 18, 21, X, and Y. Methods: A commercial quantitative fluorescent PCR kit was validated on 200 known, blinded, prenatal DNA specimens. The kit was then validated prospectively on 1069 amniotic fluid specimens, and the results were compared with the karyotype results and the results of interphase fluorescence in situ hybridization testing, when performed in the course of standard care. Turnaround time was monitored in a subset of the prospective specimens. Results: The analytical sensitivity and specificity of testing in the validation specimens were 98.9% and 100%, respectively. There were no false positives and a single false negative, a mosaic sex chromosome aneuploidy interpreted as normal. In the prospective study, the analytical sensitivity and specificity were 98% and 100%, respectively. No false positives and a single false negative, again a sex chromosome mosaic, were detected. Overall, 72.5% of all chromosomal anomalies and 87.7% of clinically significant chromosome anomalies were detected by quantitative fluorescent PCR. The average and median turnaround times were 30.5 and 25.1 hours, respectively. Conclusions: Quantitative fluorescent PCR is a robust and accurate method of rapid prenatal aneuploidy detection. Genet Med 2011:13(2):140 -147.