Mycobacterium bovis is the cause of bovine tuberculosis (bovine Tb) in animals and is considered to be zoonotic and accordingly it infects humans, although cattle are the main host. Buffalo can also be infected and develop bovine Tb. In Iran, almost half a million buffaloes are farmed, mainly in three provinces. In West Azerbaijan, which has the largest numbers of buffaloes, cattle and buffalo are often farmed together. According to the reports of the Iranian Veterinary Organization over the last 25 years, there have been no reports of bovine Tb in buffalo, although the disease is often reported in cattle in this province. Eighteen and 140 pathology specimens from cattle and buffalo, respectively, collected from West Azerbaijani abattoirs were cultured. From one buffalo specimen out of 140, M. bovis was recovered, whereas the pathogen was isolated from 13 cattle specimens. Spoligotyping showed a relatively higher polymorphism within these isolates compared with M. bovis isolated from other Iranian provinces.