Avian influenza virus subtypes H5, H7, and H9 related epidemics are accountable for huge losses to the poultry and avian industry in Pakistan. Well-timed and accurate diagnosis of subtype(s) concomitant with a specific epidemic allows a margin of prophylaxis to farmers through requisite vaccination. The current study was designed to develop and validate a rapid, accurate, and efficient diagnostic technique based on a modified strategy in reverse transcription PCR (RT-PCR) for simultaneous detection of avian influenza virus subtypes H5, H7, and H9. Instead of 3 pairs of primers, a single common forward sense primer and 3 negative sense primers specific for 3 subtypes were used. Genotyping on 2% agarose gel sorted out specific products of desired sizes for individual and mixture samples of three subtypes.