The objective of this study was to compare the positive detection rates obtained using the Oxoid IMAGEN® direct immunofluorescence assay (designated as IF) with those obtained using the CLART® PneumoVir multiplex RT-PCR DNA microarray assay (designated as RT-PCR) in the diagnosis of respiratory viruses in hospitalized children. This was a retrospective study of 62 individuals < 18 years old who had nasopharyngeal aspirates collected for virus identification in a tertiary university hospital in south Brazil between January 1, 2014 and December 31, 2014. All 62 nasopharingeal aspirates were analyzed using both assay methods. The main outcome to be measured was the difference in the proportion of test samples returning a positive virus detection result between the IF and the RT-PCR. The McNemar test was used for data analysis and the results showed that the RT-PCR and the IF methods produced 55 (88.7%) and 17 (27.4%) virus-positive samples, respectively (p < 0.001). The most prevalent virus was rhinovirus (45.5% of the RT-PCR positive samples). The RT-PCR method increased the detection rates of human respiratory syncytial virus, influenza A virus and parainfluenza 3 virus. The RT-PCR and IF had concordant results in 19 samples (30.6%) and discordant results in 43 samples (69.4%). It is concluded that in comparison to the Oxoid IMAGEN® IF method, the CLART® PneumoVir multiplex RT-PCR method had a greater potential to contribute to the clinical management of hospitalized children due its greater ability in detecting respiratory viruses than the IF method.Copyright © 2020. Published by Elsevier B.V.