Mycoplasma gallisepticum and M. synoviae are the causes of mycoplasmal infections of most concern for commercial poultry operation. These infections are very difficult to eradicate from multi age layer farms. So Science and Technology Development Fund (STDF) gave spot lighted for solving the problem by using molecular biology in preparation of Mycoplasma gallisepticum and M. synoviae recombinant vaccine to control mycoplasmosis in chickens. The Egyptian market lack the presence of this vaccine, therefore our project started with the detection of virulent genes in our field strains. These virulent genes were cloned and expressed in E.coli competent cells for preparation of recombinant proteins which used in the preparation of recombinant vaccine. A laboratory experiment followed by in field study was done for evaluation of the prepared vaccine. The vaccine succeeded in protection of the vaccinated layer flocks against Mycoplasma infection