Mycoplasma gallisepticum (MC) is a persistent, highly transmissiblechicken pathogen. It predisposes the birds to other infections such as E. coliyielding significant losses in performance and associated economics to allsectors of the poultry industry. In fact, one important feature ofMgallisepticum infection is that it can persist in the bird during all live, evenin the presence of the humoral antibodies.Therefore, the aim of this work is to perform a comparison between theconventional methods, conventional PCR and real time PCR in diagnosis ofsingle M gallisepticum infection and combined with E. coli in respiratorymanifested chickens, to apply a comparative study between natural andexperimental infections of chickens with M gallisepticum and/or E. coli andto evaluate the role of M. gallisepticum and E. coli virulence genes inoccurrence of respiratory diseases in chickens.In the present work, 204 specimens (102 lungs and 102 tracheas) of 102chickens with respiratory manifestations collected from EI-sharkia and EI-dakahlia Governorates from different breeds with different ages all over theyear seasons. The specimens were examined phenotypically andgenotypically for the detection of the most important virulence genes ofM.gallisepticum and E. coli accused of CRD occurrence in chickens.Concerning to the prevalence of single and combined infection withM.gallisepticum and E. coli in respiratory organs of respiratory manifestedbroilers, the recorded results revealed that E. coli was highly detected with apercentage of 49% followed by M gallisepticum (13.7%) and lastly combinedM. gallisepticum E. coli infection (8.8%).