【Method】The virulence of Newcastale disease virus (NDV) strains was determined by the lysis site sequences of the viral F protein. Accordingly, two pairs of primers were designed to match such sequences of virulent NDV strains and avirulent strains, respectively. Viral RNA was amplified using the two primers respectively by one-step SYBR Green I RT-PCR, and the virus was pathotypied with the amplification effects of the two pairs of primers and the Tm values of the amplification products. 【Conclusion】Pathotyping results of 8 unknown strains of this assay were as the same as those of Mean Death Time (MDT) and sequencing, which suggested that this assay could be used to pathotype NDV strains.