Three different laboratory diagnostic tests were used for rapid and accurate diagnosis and typing of different Mycoplasma strains causing respiratory diseases in cows. A total of hundred lung samples from cows, with pneumonic lesions were collected from EI- Basateen abattoir, and examined for the presence of Mycoplasma, by using isolation, polymerase chain reaction (PCR) and measuring the protein profile of the different isolates using (SDS- PAGE). The results indicted that Mycoplasma bovis showed the highest percentage among other mycoplasma isolates (M. arginin and M. bovirhinis and untyped strains). The result reported that PCR was more rapid, sensitive and specific for accurate detection of Mycoplasma infection in comparison with culture method that was more specific, most difficult and laborious of time and effort. The presence of specific protein bands at 112, 96, 65, 50, 41, 30, and 17 KDa in M. bovis isolates was characteristic. Slight differences in the expression levels of some proteins could be detected.