Mycoplasma bovis is one of the important pathogens in mycoplasma types that cause disease in cattle. The young calves from one to four months of age are most likely to develop pneumonia caused by M. bovis. In this study, we isolated M. bovis from tracheal swabs of cattle which showed respiratory symptoms. A total of about 100 tracheal swab samples were collected from cattle in Kafer El-Shikh slaughterhouse, Egypt. The collected samples from cattle were between 3-12 months of age. Mycoplasma bovis was identification in tracheal swab samples by using 16S rDNA gene sequencing and biotyping by using rep-PCR, respectively. The microbiological method could not give positive results, while the PCR showed that M. bovis infections were positive in 16 different cattle samples with about 16%. The partial sequences of the 16S rRNA genes of the Mycoplasma isolates were obtained and phylogeny tree showed that Sixteen Mycoplasma isolates were identified into Mycoplasma bovis. the similarity to Mycoplasma bovis MYC 84, M. bovis L22 and M. bovis MYC 76 was 100, 99 and 95%, respectively. The ten Rep-PCR primers produced about 139 fragments, 53.3% of them consider as monomorphic and  46.7% of them consider as polymorphic bands. According to genetic similarity and intraspecies differentiation, the sixteen Mycoplasma isolates were grouped into two main different clusters with about 60% genetic similarity in genetics dendrogram. These results suggest that PCR technique is a specific molecular detection technique identified to determine Mycoplasma and It is easy and fast methods to detect and isolate infected animals.