Use of multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza type B virus and influenza A virus subtypes H5N1, H3N2, and H1N1 has been described. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2) copies per microliter or 10(-3)-10(-2) TCID50/L for each subtype, as well as a high reproducibility with coefficient of variation (CV) ranging from 0.27% to 4.20%. The assays can be performed commendably on various models of real-time PCR instruments; including ABI7500, ROCH 2.0, and Mx3005p. In an analysis of 436 clinical samples from patients during the year 2009, this detection method has successfully identified 261 positive samples, as compared to only 189 positive samples using the conventional cell culture systems, and at the same time further differentiated them as 35 type B, 21 subtype H1N1, and 205 subtype H3N2. The results indicate that the multiplex real-time RT-PCR method is a potential tool for rapid screening of influenza virus from a large pool of clinical samples during flu pandemics and facilitates early influenza virus identification in most public health laboratories around the world.