Transfer of influenza A viruses from animal hosts to man may lead to the emergence of new human pandemic strains. The early detection and identification of such events are therefore paramount in the surveillance of influenza viruses. To detect and partially characterize influenza A viruses from different animal species, a combined reverse transcription (RT)-PCR heteroduplex mobility assay (HMA) was designed. This M gene RT-PCR was shown to be sensitive and specific for the detection of human, avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine viruses of 15 different subtypes, with between 1.9 and 21.4% nucleotide divergence, were differentiated by HMA. Sequencing of the amplicons showed that the heteroduplex mobility patterns correlated with the sequence divergence between test and reference DNA. The application of the RT-PCR HMA method for rapid screening of samples was assessed with a reference panel of viruses of human, avian, and swine origin. The avian H9N2 virus A/HongKong/1073/99, which crossed the species barrier to humans, was screened against the reference panel. It was found to be most closely related to the avian A/Quail/HongKong/G1/97 H9N2 reference PCR product. Sequence analysis showed a nucleotide divergence of 1.1% between the A/Quail/HongKong/G1/97 and A/HongKong/1073/99 amplicons. From the results of our work, we consider the RT-PCR HMA method described to offer a rapid and sensitive means for screening for novel or unusual influenza viruses.