Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis.