AIMS: To evaluate the feasibility of identifying viruses from aircraft cabin air, we evaluated whether respiratory viruses trapped by commercial aircraft air filters can be extracted and detected using a multiplex PCR, bead-based assay. METHODS AND RESULTS: The ResPlex II assay was first tested for its ability to detect inactivated viruses applied to new filter material; all 18 applications of virus at a high concentration were detected. The ResPlex II assay was then used to test for 18 respiratory viruses on 48 used air filter samples from commercial aircraft. Three samples tested positive for viruses, and three viruses were detected: rhinovirus, influenza A and influenza B. For 33 of 48 samples, internal PCR controls performed suboptimally, suggesting sample matrix effect. CONCLUSION: In some cases, influenza and rhinovirus RNA can be detected on aircraft air filters, even more than 10 days after the filters were removed from aircraft. SIGNIFICANCE AND IMPACT OF THE STUDY: With protocol modifications to overcome PCR inhibition, air filter sampling and the ResPlex II assay could be used to characterize viruses in aircraft cabin air. Information about viruses in aircraft could support public health measures to reduce disease transmission within aircraft and between cities.