A simplified but reliable method was developed for the polymerase chain reaction (PCR)-based detection of genetically modified (GM) plants. The modified CTAB (mCTAB) method enabled us to prepare a high quality of genomic DNA from several hundred plant leaf samples in one day. Using DNA samples prepared from seven dicots and two monocots, approximately 1.75-kb regions spanning 17 S to 25 S ribosomal RNA genes were successfully amplified in a 2X PCR pre-mix containing BLOTTO. Further fidelity assessment of the mCTAB method by PCR analysis with Roundup Ready soybean (RRS) and non-RRS plants showed that the DNA samples prepared alternately from each of two lines were evidently free of cross-contamination. These results demonstrate that the mCTAB method is highly recommended for the rapid detection of transgenes in large numbers of leaf samples from diverse transgenic plants.