A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCyclerTM system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCyclerTM software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.