Since the pandemic influenza A (H1N1) 2009 ((H1N1)pdm09) virus spread all over the world, the (H1N1)pdm09 virus has been circulating with seasonal influenza viruses. We developed rapid and sensitive one-step multiplex real-time RT-PCR assays (rRT-PCR) for simultaneous detection of influenza viruses currently circulating in humans, and the avian A/H5 virus. The detection limit of each assay was 4.8 to 1 copies per reaction and no cross-reactivity with other major respiratory pathogens was found. Analytical positive predictive value (PPV), negative predictive value (NPV) sensitivity and specificity were 100%, 94.1%, 93.7% and 100%, respectively. Clinical evaluation revealed that 1,976 (16.5%) of 11,963 throat swabs from patients with respiratory symptoms were confirmed as 1,651 (83.6%) A/H1pdm09, 308 (15.6%) A/H3 and 17 (0.8%) B virus during the 2010-2011 influenza season. Collectively, the multiplex rRT-PCR assays described here provide a practical tool for reliable implementation of influenza surveillance and diagnosis.