We describe a highly accurate method for determining the sex of human embryos via real-time polymerase chain reaction (PCR) amplification of highly-conserved, moderately-repeated sequences within the TSPY genes on the Y chromosome and the U2 genes on chromosome 17. Individual male lymphocytes, female lymphocytes, and blastomeres from donated cleavage-stage embryos were lysed prior to PCR using an optimized buffer containing proteinase K. Molecular beacons, a new type of fluorescent probe, were used to detect and quantify accumulating amplicons during each cycle of PCR carried out in closed tubes. The present work is part of an ongoing study to construct and implement a new, convenient and reliable system of preimplantation genetic diagnosis (PGD).