In this study the prevalence of Mycoplasma species was studied in diseased turkey flocks showed respiratory symptoms, sinusitis, lameness as well as decrease in egg production in Al Minia and Sohag Governorates by both culturing and serological methods. Six field isolates were tested by PCR and compared with the standard M. gallisepticum reference (R) strain. All the examined field isolates were identified as M. gallisepticum. RAPD-PCR was used as technique to differentiate between the strains of the same Mycoplasma species. DNA profiles of five M. gallisepticum field isolates were compared with that of M. gallisepticum reference strains (F and R) using fan primer. The banding patterns of that isolates were highly similar to those of M. gallisepticum reference strains. The in vitro evaluation of antimicrobial activities using the minimum inhibitory concentrations (MICs) test against the isolated Mycoplasmas proved that the tilmicosin and tylosin possessed the lowest MICs compared with other six antimicrobials, so it is recommended to be used for in-vivo treatment and also they are effective in eradication programmes of field Mycoplasma infection in poultry. In conclusion, M. gallisepticum is the predominant cause of turkey respiratory manifestations. RAPD-PCR technique detects the genetic diversity in the natural populations among field isolates. Tilmicosin and tylosin had the lowest MICs than other antimicrobials used in this study against avian M. gallisepticum.