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Multiplex assays of more than one viral gene target in a single tube have the advantage of rapid screening of a large number of potential viral pathogens in a short time. A multiplex real\u2010time PCR assay was used in this study for detection of respiratory RNA and DNA viral infections in 728 specimens received from 585 adult and pediatric patients comprised of symptomatic and asymptomatic organ transplant recipients and non\u2010recipients for diagnosis of respiratory illnesses and for routine clinical monitoring. Multiplex PCR was more sensitive than the multiplex immunofluoresence culture assay (R\u2010mix) and also detected additional respiratory viruses that were not covered by the R\u2010mix panel. The number of respiratory viruses detected in symptomatic patients was significantly higher than asymptomatic patients in both adult and pediatric patients. Herpesviral infections were the predominant cause of lower respiratory tract infection in the organ transplant recipients, whereas respiratory syncytial virus was the most common pathogen in non\u2010transplant patients particularly children. Multiplex real\u2010time PCR for detection of respiratory viruses has the potential for rapid identification of viral pathogens. In this era of emerging viral infections, addition of newer viral targets to the multiplex PCR panels will be beneficial in determining both patient management and public health epidemiology. J. Med. 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