The aim of this study was to develop a rapid, cost-saving triple reverse transcription polymerase chain reaction (triple RT-PCR) for subtyping H9N2 avian influenza viruses (AIVs). The three primer pairs for amplification of target sequences of nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA) genes, respectively, were designed for subtyping the viruses in the triple RT-PCR. The sensitivity of triple RT-PCR was found to be 10(2) copies per reaction for each of NP, H9 and N2 gene. The specificity tests indicated that all of NP, HA and NA genes were positive for H9N2, only NP gene was positive for H5N1 and H1N1 AIVs, and the results were negative for the other avian viruses including Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, duck hepatitis virus and avian encephalomyelitis virus. A total of 112 clinical samples were evaluated by the assay and the results showed that the sensitivity and specificity of triple RT-PCR were in accordance with the virus isolation. In conclusion, this method is rapid and cost-effective making it feasible and attractive for large-scale epidemiological investigation of H9N2 influenza virus.