To study the incidence of bovine tuberculosis in buffaloe and cattle in abattoirs of Peshawar, Khyber Pakhtunkhwa, The suspected lung and liver samples were collected at random from buffaloe (Bubalus bubalis) and cattle (Bos taurus) from abattoirs of the city and were analyzed in the laboratory of Veterinary Research Institute (VRI) for the presence of Mycobacterium bovis through acid fast staining, culture and polymerase chain reaction (PCR). A total of 151 animals including 121 buffaloe and 30 cattle samples were analyzed in this study from July to November 2011. Seventeen (14.04%) of the 120 buffaloe were found positive for AFB, while the remaining one hundred and four buffaloe (85.9%) were negative. Amongst the cattle only four were positive out of 30 (13.33%) for bovine tuberculosis. Out of the total 151 animals, 302 samples were collected, one each from lung and liver respectively and 27 (8.94%) were found with AFB. It was observed that 13.90% of the lung tissue were AFB positive while 6 (3.97%) out of 151 liver samples showed the AFB incidence. The AFB culture technique was effective in detecting the presence of Mycobacteria, as 8.94% of the overall suspected tissue samples were found AFB positive. The detailed laboratory examinations such as, PCR and other biochemical tests to supplement the cultural identification are recommended for accurate and reliable identification of bovine tuberculosis among the cattle. The microscopic examination can only determine the acid fast organisms in the suspected samples whereas culture on stone brink media along with biochemical and PCR techniques can differentiate between acid fast Mycobacterial species.