The present study was performed to detect and isolate the Mycoplasma as causative agent of atypical pneumonia in feedlot lambs. Samples (180) were collected from lambs suffering clinically from pneumonia. During the period October 2013 to March 2014. These samples were included nasal swabs (60), tracheal swabs (60) and infected lungs (60). Samples were cultured in the selective media (Mycoplasma Broth Base) and incubated aerobically at 35C° for 4 – 14 days. When turbidity appeared inoculum of each broth was cultured on Mycoplasma Glucose Agar medium and incubated at 35C° for 7 -14 days in candle jar. Primary isolation was demonstrated by color changing of diphasic media, The ability of staining with Dennis stain, reduction of Tetrazolium, red blood cell lysis and fried egg appearance of Mycoplasma colonies was also studied. The results of present study also showed the total rate of Mycoplasma isolation (49.4%) from total samples, and the highest rate of isolation was from Nasal swabs (58%). While, the rate of isolation from Tracheal swabs (55.3%) and from infected lungs (35%).