Four different models of infection of chickens with Mycoplasma gallisepticum (MG) were evaluated. Normal or immunosuppressed chicks (10 days old) were infected with the MG R strain via eye and nasal drops or by direct air sac injection. Bacterial load in the lungs was quantified and air sac and tracheal lesion scores, tracheal mucosal thickness and humoral immune responses were assessed. Serum antibody responses were assessed by use of a serum plate agglutination test. Three days post infection (dpi), all immunosuppressed chicks had developed significant respiratory signs. Chicks infected via air sac injection had significant differences in serum antibody and gross lesion scores at 5 dpi. All chicks had developed pathological changes by 7 dpi. Air sac inoculation of immunosuppressed chicks produced more significant (P ≤0.05) lesions, and these birds had the highest bacterial load in the lungs compared with other groups.