Mycoplasma gallisepticum (MG) is responsible for serious economic losses in poultry industry in Pakistan. The present study was designed to evaluate the molecular and immunological cross reactivity among MG isolates recovered from broiler birds. For this purpose a total of four hundred and thirty-four (n=434) specimen samples were taken from suspected broilers as well as environmental samples of forty commercial poultry (broiler) farms from DIK, Tank, Peshawar, Abbottabad and Mansehra districts of Khyber Pakhtunkhwa. These samples were inoculated and serially passage into Modified Frey’s broth. One hundred and nine (25.1%) samples showed turbidity and were further processed through biochemical tests (Catalase production, Tetrazolium reduction, Phosphatase production, Film and spot Assay and Glucose fermentation). Typical fried egg colonies on Frey’s agar were purified. Biochemical tests indicated Mycoplasma species as 2.5% of total specimen samples. The PCR results confirmed (185 bp) three MG isolates (MG-I, MG-II and MG-III) with 0.7% prevalence in total specimen samples. All three antigens (Formalin inactivated and Montanide adjuvanted) were subcutaneously inoculated @ 0.5 ml into rabbits and booster on 7 days post first inoculation. Sera samples collected after 14 days post booster shot, were processed for antibody titration using Haemagglutination inhibition (HI) test with homologous and heterologous antigens. The immunological cross reactivity indicated all three MG isolates as distinctly separate from each other. The immunological cross reactivity was existing with 66 to 87.18% among all three isolates. The results showed high homologous GMT titers as compared to heterologous for each MG isolate.