ABSTRACT   Respiratory infections are the most dangerous diseases facing poultry industry. Ornithobacteriosis is a respiratory disease in chicken broiler farms, it causes severe economic losses (high condemnation rate at slaughter, high treatment costs). Scientific research papers should be focused on the causative agent in order to make a plan for the ideal steps for its isolation and identification, in addition to prevention and control of this infection. The present work was conducted to isolate and identify Ornithobacteriumrhinotracheale (ORT) in chicken broilers.A total of 195 samples (trachea, lungs and air sacs) were collected from dead and sacrificed broiler chickens aged (20 - 42 days old) from different broiler chickens farms in Assiut and New Valley Governorates for possible recovery of ORT. Samples were cultured on blood agar plates under 7.5 – 10 % CO₂ tension at 37ºC for 48 hours. Bacteriological examination used for isolation and identification of the ORT bacteria (cellular and colonial morphology, biochemical reactions, API 20 NE and growth on MacConkey's agar) showed 32 suspected isolates at percentage of 16.4%. Results of isolation by conventional method were confirmed by molecular identification using Polymerase Chain reaction technique (PCR); which revealed the positive detection of only 5 strains of ORT and produced the predicated 625 bp amplification product. Serotyping of the isolates by using agar gel precipitation test proved that all isolates belonged to serotype A. Challenge of broilers aged 14 days old with ORT strain alone or combined with live ND vaccine by spraying method caused mild respiratory manifestations with tracheitis, pneumonia and mild airsaculitis. While Infection of chicks with ORT via spray combined with prior administration of ND vaccine (stress factor) caused a moderate respiratory signs and more severe lesions.  From the results obtained in this work, we can conclude that:  The prevalence of ORT infection in broiler chickens in Assiut and New Valley governorates was 2.56% from living and scarified birds. The current investigation revealed that the isolates of ORT bacteria belonged to serotype A. Post mortem lesions especially yogurt like exudate in the abdominal air sac considered very pathognomonic in the tentative diagnosis of ORT infection. While PCR technique was proved to be more sensitive and specific for detection of ORT pathogen.