A total of 94 chicken samples were examined for Mycoplasma gallisepticum and Mycoplasma synoviae either by culturing procedure or by using the multiplex PCR. The comparative analysis of the obtained results indicated that the multiplex PCR was more sensitive and reliable in the detection of both organisms, where the multiplex PCR detected Mycoplasma gallisepticum in 20.21% of the tested samples while it gave 5.319% prevalence for Mycoplasma synoviae. Moreover isolation and culturing procedures detected Mycoplasma gallisepticum and Mycoplasma synoviae in a prevalence rate of 15.956% and 2.127% of the tested samples comparatively. So these results concluded that, the multiplex PCR has become a valuable and easy test aiding in diagnosis of M. gallisepticum and M. synoviae infection in chickens.