Bovine tuberculosis is a chronic bacterial disease of animals and humans caused by M. bovis. In a large number of countries, bovine tuberculosis is a major infectious disease among cattle and other domesticated animals. M. bovis can infect humans, primarily by the ingestion of unpasteurized dairy products but also in aerosols and through breaks in the skin. Raw or undercooked meat can also be a source of the infection. The diagnosis is confirmed by the isolation and identification of M. bovis on selective culture media or by different polymerase chain reaction (PCR) assays. In the current study: A total of 49 specimens were collected from four major abattoirs (El-Basateen, El-Monieb, Beni-Suef and Al-Fayoum) to be analyzed bacteriologically for: isolation, identification and confirmation of M. bovis with molecular methods. Only 19 isolates were found to be positive slow-growers Mycobacterium species by conventional cultivation method on solid medium (LowensteinJensen medium). Genotyping detection of MTC by amplification of gene responsible for production of MPB70 secretory protein by conventional PCR and by amplification of extRD9 region by real-time PCR was carried out directly on specimens. Out of 49 DNA templates extracted directly from specimens, 14 specimens were confirmed to be infected by MTC by conventional PCR and 31 specimens were confirmed to be infected by MTC by real-time PCR. The results revealed that real-time PCR assay was the most sensitive, powerful and efficient assay compared with conventional PCR.