Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB), primarily in bovines; it has a wide host range and zoonotic features. In this report, we aimed to detect the antibodies specific against M. bovis in large ruminants using P22 ELISA, geographically distribute bTB-infected cattle and buffalo cases using single intradermal tuberculin (SIT) in Sohag governorate, and to carry out comprehensive and phylogenetic genome analysis to study the drug resistance genes of M. bovis strains in Egypt. Using P22 ELISA was applied in large ruminants to detect M. bovis specific antibodies in serum samples of cattle and buffaloes; no positive cases were detected in either cattle or buffalo. Based on SIT, no confirmed cases were detected from 2020 to 2023. In 2024, 24 positive cases (0.07%) and 37 positive cases (0.2%) were confirmed in Sohag Center and Akhmim respectively. Using the PATRIC server on 11 M. bovis isolates, all isolates were susceptible to Streptomycin, Amikacin, Kanamycin, Capreomycin, Rifampin, Ethambutol, Isoniazid, and Ofloxacin, antibiotics, two resistant genes were identified in MBE12 and MBE7, with a monoresistance identified in MBE12 against Ethambutol antibiotic. MBE7 strain encodes OxyR resistant gene. However, more analysis is needed to understand M. bovis increased incidence, genomic diversities, and resistance in Sohag and other Governorates in Egypt.