Ornythobacterium rhinotracheale (ORT) is an emerging bacterial pathogen in poultry industry. The disease is acute highly contagious, affecting chickens and turkeys. The clinical signs, duration of the disease and mortality are extremely variable and are influenced by suboptimal environmental condition. Due to the difficulties in isolation and biochemical characterization of the bacteria, development of a fast and reliable method for diagnosis of the disease and characterization of the isolate is necessary. A combination of conventional bacterial isolation technique and colony PCR method were developed as a diagnostic tool for diagnosis of ORT infection in poultry.