Avian influenza and Newcastle disease are the highly contagious and most economically important diseases in poultry industry throughout the world. A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for the rapid and specific discrimination of H5 and H9 subtypes of avian influenza viruses (AIV) and Newcastle disease virus (NDV). Three sets of specific primers were applied in the assay based on the sequences of the hemagglutinin gene of H5-AIV, H9-AIV and fusion protein gene of NDV. 59 clinical samples including the throat washes, oral swabs, and cloacal scrapings were detected by mRT-PCR and single RT-PCR (sRT-PCR), respectively. The results indicated that the sensitivity and specificity of mRT-PCR were in accordance with sRT-PCR. The mRT-PCR developed in this study may therefore provide a new avenue to rapid detection of these important pathogens in one reaction.