Background: Respiratory tract infections are majorpublic health threats, and the identification of theircausative microbes helps clinicians to initiate timelyand appropriate antimicrobial therapy and prevent thesecondary spread of infection. The main goal of thisstudy was to compare two multiplex real-time polymerasechain reaction (PCR) assays used to detectrespiratory viral pathogens in nasopharyngeal swabspecimens. Methods: Between September and October 2017, atotal of 84 nasopharyngeal specimens were obtainedconsecutively from patients in a tertiary hospital usinga flocked swab with 3 mL universal transport medium(COPAN Diagnostics, USA). A total of 64 positiveand 20 negative sample results from the LGAdvanSure RV real-time RT-PCR kit (LG LifeSciences, Korea) were further retested using a newAdvanSure RV-plus a real-time RT-PCR kit to comparetheir performance. Results: Statistical analysis of positive and negativeagreement between the two different kits was conductedbetween the newly introduced AdvanSureRV-plus real-time RT-PCR kit and the AdvanSure RVreal-time RT-PCR. The overall agreement was 96.4%,with positive agreement of 98.4% and negativeagreement of 90%. The evaluated sensitivity andspecificity of AdvanSure RV-plus real-time RT-PCRwere 96.9% and 94.7%, respectively, with a kappavalue of 0.9 (P<0.001). Conclusion: The performances of LG AdvanSure RVreal-time RT-PCR and the new AdvanSure RV-plusreal-time RT-PCR kit showed strong overall agreement. AdvanSure RV-plus real-time RT-PCR had a betterdetection rate and could detect coronavirus 229Eand enterovirus, especially with a high detection ratein coinfection. AdvanSure RV-plus real-time RT-PCRcan be considered a useful tool for respiratory virusdiagnosis in clinical laboratories.