In this study, we report that the development of a multiplex PCR method using species-specific primers for the simultaneous detection of Poaceae family members, including adlay, barley, maize, rice and wheat, based on the sequence polymorphism of the DNA-directed RNA polymerase beta'' chain (rpoC2) genes Species-specific primers were constructed with common forward primer and each reverse primers which have differences on the basis of sequences. Each primer pairs could amplify PCR products of 443 bp for rice, 346 bp for barley, 278 bp for adlay, 221 bp for wheat and 96 for maize, respectively, from the five chloroplast DNAs. The series of template DNA concentrations were identified by the sensitivity of multiplex PCR. The band of products were clearly amplified from the DNA concentration range of 0.01 to 10 ng/μL. In addition, the species-specific primers were examined for the detection of seven commercial flour mixed products. The combination of the sensitivity of a multiplex PCR with the specificity of the primers for the detection of species would allow to be applied in analyses of processed foods.