The prevalence of Mycoplasma species from diseased chickens from different poultry farms and individual breeders in Egypt (Sohag, Assiut and Cairo Governorates) was studied by both culturing and serological methods. A total of three hundred and twenty five samples including swabs from tracheal, nasal cleft and conjunctiva as well as 325 serum samples were collected from chickens showing respiratory manifestations. These birds were of different ages (four weeks - two months) and of different breeds (native and leghorn). Mycoplasma species were biochemically identified as M. gallisepticum or M. pullorum in 220/325 swab samples with the percentage of 68%, M. gallinarum was isolated from 20/325 with the percentage 6.2% and M. arginini was isolated from 15/325 with the percentage 4.6%. By serological identification of the Mycoplasma spp. using growth inhibition test, M. gallisepticum in 148/255 with the percentage of 58%, M. pullorum in 77/255 with the percentage of 30.2% and M. gallinarum in 30/255 with the percentage of 11.8% were founded. The serological identification of Mycoplsama spp. using serum plate agglutination test proved that 277/325 were M. gallisepticum with a percentage of 85.2 % and 48/325 were M. synoviae with the percentage of 14.8%. Four field isolates were tested by PCR and compared with standard M. gallisepticum referance strains (F, PG31 and S6). All of the examined field isolates were identified as M. gallisepticum (gave characteristic 330 bp fragment), while M. pullorum and M. gallinarum were not amplified by these primers. RAPD-PCR is a more recent technique used for differentiation among different strains of the same Mycoplasma species. Also, it is a reproducible method for comparing the Mycoplasma field isolates in epidemiological studies. The technique detects the genetic diversity in natural populations among field isolates. DNA profiles of 4 M. gallisepticum field isolates were compared with that of M. gallisepticum reference strains (F, PG31 and S6) using Geary 1 and Geary 2 primers. Using Geary (1) primer, it was clear that RAPD fingerprinting is capable of distinguishing the M. gallisepticum field strains from vaccinal strains. On the other hands, It was interested to note that Geary (2) primer was not sufficient to differentiate M. gallisepticum strains (reference and field). SDS-PAGE was used for differentiation between M. gallisepticum standard and field strains. In the present study, comparison of the protein profiles of standard and field strains M. gallisepticum whole cells was carried out for detection of similarity and /or variations among the compared strains. Each protein analysis of M. gallisepticum reference strains (PG31. F, S6 and R strains) and 10 of the field isolates yielded a characteristic profile banding ranged from (18-25) bands. From the results of SDS-PAGE analysis, it was clear that most isolates were related to S6 and R reference strains.