We assess the use of high-resolution melting (HRM) analysis for rapid identification of influenza A virus subtype and detection of newly emerging virus variant. The viral matrix gene was amplified by LightCycler real-time RT-PCR in the presence of LCGreen I fluorescent dye. Upon optimization of assay conditions, all the major influenza A virus subtypes including H1N1, H3N2, H5N1, H7N3, and H9N2 were amplifiable by this method, with PCR product length of 179-bp. Real-time RT-PCR of the in vitro transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient = 0.9935) across at least 8 logs of RNA concentrations and a detection limit of 10(3) copies viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified via the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the tested and reference viral isolate. The melting profiles were consistent with minimal inter-assay variability. Hence, a HRM database and a working protocol were established for identifying these 5 influenza A virus subtypes. When tested with 21 clinical influenza A virus isolates, the results were comparable with those obtained by RT-PCR using hemagglutinin-specific primer sets. Sequence variants of the clinical isolates (n=4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.