The cause of bovine tuberculosis in cattle mainly is Mycobacterium bovis which is pathogenic for a large number of other animals, and its transmission to human constitutes a public health problem. The control and eradication of Mycobacterium bovis in Egypt depend on developing rapid and accurate detection techniques. Control of bovine tuberculosis in Egypt is mainly depending on test and slaughter policy which consume the economy and yet there is no evidence of reducing the prevalence of tuberculosis. We need rapid and accurate method for identification of M.bovis. The aim of this study, establishment of multiplex PCR for Identification of Mycobacterium bovis isolates in Egypt and comparing it with the Conventional gold standard mycobacterial culture techniques.