In this study, M. gallisepticum and M. synoviae were isolated from different breeds of chickens in Hail region of Saudi Arabia. Out of 200 samples (trachea, lungs, air sacs and synovial swabs) taken from native chickens of different ages, 100 pure mycoplasma isolates were recovered. When typed by morphological, biochemical and serological methods, 39 isolates were identified as M. synoviae, 31 isolates were identified as M. gallisepticum..Of 60 samples from Lohmman layers, 11 pure isolates were obtained and 6 of them were identified as M. synoviae, 1 was M. gallisepticum. Of 140 samples collected from Ross chickens and breeders, only samples collected from breeders aging 8-14 weeks (40 samples) resulted in positive mycoplasma isolation. Fifteen pure mycoplasma isolates were identified as M. gallisepticum, 4 were identified as M. synoviae. recovered of which 3 were Tracheal samples and synovial fluids resulted in the highest isolation rates from native and Lohmman chickens, respectively while synovial fluid and tracheal samples resulted in equal rates of Mycoplasma isolation from Ross chickens. Agar media containing tryptone soy agar base resulted in bigger Mycoplasma colonies and nipples.