Newcastle Disease (ND) is highly contagious infection of poultry that causes nervous signs and mortality in poultry. This study has been one during two years from 2010-2011 in Shiraz, Iran. The poultry industry in Fars province faced an almost heavy loss that was characterized by mild and in some flocks high mortality and respiratory distress. The aim of this study was designed to clarify the roles of the Newcastle Disease Virus (NDV) by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) assay and Mean Death Time (MDT) test in recent outbreak in Fars province in Iran. In virology tests, Newcastle Disease Virus (NDV) was isolated from tracheal and cloacal swabs samples. Diagnosis implies the differentiation to virulent and non-virulent Newcastle disease virus. During the period of this study a total 30 commercial broiler flocks with high mortality in Fras province were visited. Samples were collected from chickens with respiratory distress. Two oligonucleotide primers, representing the sequence at the cleavage site of the F protein of both virulent and non-virulent NDV strains, respectively, were used to differentiate NDV. Using the RT-PCR was able to differentiation 6 NDV reference strains 6 of which were virulent.