In order to study the Molecular characterization of mycoplasma isolated from chicken. A total number of  200 samples collected from  birds showing  respiratory manifestations and apparently healthy chicken of  different ages(two weeks: two months) from different localities(al-ismailia & sharkeya Governorates). These samples included 110 samples from respiratory organs(trachea -lung -air sac) & 75 swabs from nasal cleft and 15 samples from  fluid of swollen joints. A trial for isolation and identification of different Mycoplasma was done using conventional and recent techniques. All the results were finally confirmed by polymerase chain reaction (PCR) which showed products of the amplified16S rRNA gene and mgc2 gene of  MG, detection of 16s rRNA gene of MS and 16S rRNA gene of un typed Mycoplasma in chickens using a set of primers tested and compared according to reference strain .The PCR amplification products were electrophoresed on 1% agrose gel stained with ethidium bromide. All the examined field isolates were identified as Mycoplasma (gave a characteristic common band at different levels of bp fragment). Primary isolation of the microorganism on PPLO medium, which appeared as fried egg when examined by dissecting microscope yielded118 positive samples with a total incidence rate 59%. The highest recovery rate was from respiratory organs (72.7%) followed by swabs from nasal claft (46.7%)  and samples from swollen joints (20%).Sequence analysis of two Mycoplasma gallisepticum (strains for mgc2 gene from chicken revealed high similarity with the homologous reference strains on Gene Bank. and take these  experessions on Gene Bank as Man-Reh.1/Mg/CK/EG016 acc# KY404986  and Man-Reh.2/Mg/CK/EG016 acc# KY404987.