In most countries, tuberculosis caused by Mycobacterium bovis is mainly a disease of cattle but can infect buffalos too. The disease can be controlled successfully by mean of a test-and-slaughter program. In Iran test-and-slaughter program has started since 1971 and prevalence of bovine tuberculosis reduces from 5% to less than 0.12% in recent years. In Western Azarbaijan, North West of Iran, the prevalence of bovine tuberculosis is 0.06%. Tracing the source of infection and finding the animal reservoirs is one of the important parts of an eradication program against bovine tuberculosis and that can be achieved by differentiation of M. bovis isolates. Molecular typing techniques are powerful tools for epidemiological investigations and have been extensively used for this purpose. To understand the molecular epidemiology of bovine tuberculosis in the region and possible role of buffalos, 140 specimens collected from buffaloes at abattoirs bacteriologically were cultured for Mycobacteria. Only one specimen was positive in culture. The isolate was identified as M. tuberculosis complex by conventional biochemical tests and PCR targeting the insertion sequence IS6110. Restriction fragment length polymorphism (RFLP) analysis with polymorphic GC rich repeat (PGRS) and direct repeat (DR) probes showed that this isolate is distinct from M. bovis BCG and M. bovis isolates (n=10) isolated from cattle at different provinces. This is the first case of infection with M. Tuberculosis complex in buffalos in Iran. The role of buffalos in transmission of infection to humans and animals needs to be investigated.