We developed a multiplex polymerase chain reaction (PCR)-multiplex ligase chain reaction (LCR) (MPCR-MLCR) technique as a novel approach for the simultaneous detection of recombinant DNA segments (e.g., promoters, trait genes, and terminators) of genetically modified (GM) crops. With this technique, target DNA regions were amplified by multiplex PCR, the PCR products were then subjected to multiplex LCR as template DNAs, and the LCR products were then analyzed by polyacrylamide gel electrophoresis and subsequent fluorescent scanning. Seven recombinant DNA segments commonly introduced into some GM crop lines were selected as target DNA regions. In addition, another MPCR-MLCR system for the simultaneous detection of three endogenous DNA segments was designed as a positive control test. The specificity and sensitivity of the method were examined. The method allowed us to detect GM crops comprehensively and is expected to be utilized for efficient screening of GM crops into which any one of the seven recombinant DNA segments have been introduced, and for profiling the segments.