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Bovine tuberculosis commonly called TB, is caused by bacilli Mycobacterium bovis (M. bovis), a potential zoonotic pathogen that seems to cause direct economic losses other than single microbial agent on livestock and indirect impact on human health. An investigation of bovine tuberculosis in buffaloes was carried out in Hyderabad and Tando Allahyar districts to determine the prevalence of bovine tuberculosis. The buffaloes (n=120), were first screened through single intradermal tuberculin test (SITT) and both positive and negative reactors of SITT were used for enzyme linked immunosorbent assay (ELISA). Attempts were further made to isolate the M. bovis organism from the milk samples using traditional culture test. Overall prevalence of 4.16%, 8.33% and 2.5% was recorded by SITT, ELISA and culture test respectively. A somewhat higher prevalence was recorded in Tando Allahyar district (SITT 6.66%; ELISA 10%; culture test 5%) as compared to Hyderabad district (SITT 1.66%; ELISA 6.66%; culture test 0%). The statistical analysis did not showed any association (P>0.05) of herd size, sex, age, milk yield and type of farming with the disease, regardless of district, whereas, sex, age and milk yield showed significant (P<0.05) association with the disease through SITT in Tando Allayer district. This study concluded that bovine tuberculosis is present in apparently healthy buffalo herds of Hyderabad and Tando Allayer districts. Moreover, infected animals shed the M. bovis pathogen in milk that could be a potential hazard to public health.