We developed the initial real-time reverse transcription PCR assay system for seasonal influenza viruses in 2011. This prototype assay system could detect and identify specific influenza A virus subtypes[H1N1, H3N2 and (H1N1) pdm09] and influenza B virus. In the 2012-2013 season, our prototype PCR assay didn't work well because of point mutations occurred in the neuraminidase (NA) gene of the A (H3N2) strain. We improved the prototype assay by changing the target gene for A (H3N2) strain (2013 improved PCR assay). Moreover, we added the measurement system for the matrix (M) gene that was well conserved and common to all influenza A subtypes. In the 2013-2014 season, point mutations in the hemagglutinin (HA) gene of the A (H1N1) pdm09 strain lowered the sensitivity of the 2013 improved PCR assay, so that we changed the target gene for A (H1N1)pdm09 strain (2014 improved PCR assay). We analyzed swab samples from 1,721 patients in total by at least one of the three PCR assays we developed, and demonstrated that the PCR assays had excellent sensitivity and specificity compared with those of the commercially available rapid immunochromatography kit we used. In this study, the M gene was positive in all patients who were finally diagnosed as influenza A positive by 2013 or 2014 improved PCR assay. Therefore, measurement of the M gene, which is hardly to be affected by antigenic drift of influenza viruses, is thought to be useful in clinical practice.