A cross-sectional study was conducted at Butajira Municipality abattoir from December, 2009 to April 2010 to investigate the epidemiology of bovine tuberculosis (bTB) in Butajira, Southern Ethiopia. Postmortem examination, mycobacterial culturing and multiplex genus typing techniques were used. An overall prevalence of 9% (40/446) of the animals examined harbor gross tuberculous lesions up on detailed post-mortem examination. Statistically significant difference was observed in the prevalence of bTB between different age groups (χ 2 = 11.441, p= 0.003) and body condition scoring (χ 2 = 10.384, p = 0.006). Higher prevalence of bTB was observed in older animals and animals with poor body condition. Bacteriological culture of the 40 samples gave growth on 13 with 9 of them acid fast Bacilli (AFB) positive. Genus typing of the AFB positive isolates by multiplex polymerase chain reaction (m-PCR) revealed seven non-tuberculous mycobacterium (NTM) and 1 Mycobacterium tuberculosis complex (MTBC) isolates. Further characterization of the isolates at specific species and investigation of the disease is recommended for controlling it in livestock and safeguard public health.