An investigation in buffaloes was carried out in Hyderabad and Tando Allahyar districts, Sindh province, Pakistan to determine the prevalence of bovine tuberculosis. The buffaloes (n=120) were first screened through single intradermal tuberculin test (SITT), then their sera were used for enzyme-linked immunosorbent assay (ELISA). Attempts were further made to isolate the Mycobacterium 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%). Statistical analysis did not show any association (P>0.05) of herd size, sex, age, milk yield and farming type with the prevalence of the disease. Whereas, SITT showed a significant (P<0.05) association of sex, age and milk yield with the prevalence of the disease in Tando Allayer district. The results of present study revealed 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.