A cross sectional study was conducted from December 2014 to May 2015 in Gambella town municipal abattoir and health centers to investigate the prevalence of BTB, isolation and molecular characterization of its causative agents and to assess its public health implications in Gambella, Ethiopia. Postmortem examination, bacteriological culturing, RD deletion typing and spoligotyping were used for investigation. The overall prevalence of BTB in cattle was 13.2% (66/500) on the basis of detailed postmortem examination. Statistical significant difference was observed in the prevalence of BTB among different body conditioned animals (χ2 = 39.105, P=0.000and breeds (χ2 = 24.996, P=0.000). Molecular characterization of 11 mycobacterial isolates from human patients using RD9 deletion typing showed that all were M. tuberculosis and further spoligotyping of the isolated revealed that SIT289, SIT134, SIT1634, SIT142 and one new strain not found in the spoligotype databases.