•SARS-CoV-2 RNA detection is performed for clinical diagnosis of COVID-19.•The detection of two different targets on the SARS-CoV-2 genome is recommended.•An internal control is required for evaluating the assay qualities.•A multiplex rRT-PCR methodology was developed for SARS-CoV-2 RNA detection.•A multiplex rRT-PCR will enable reducing reagent use and cost, and handling time. The detection of SARS-CoV-2 RNA by real-time reverse transcription–polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.