Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50-100 copies/mL and 5-100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.