Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log 10 dynamic range, a reproducible (R 2 > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%– 98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.