Automated sequence analysis of a 410-bp region of the axyR gene in 105 Mycobacterium tuberculosis complex isolates identified a polymorphic nucleotide that differentiated Mycobacterium bovis isolates from other complex members. All 29 M. bovis isolates sequenced had an adenine residue at nucleotide 285, whereas all 76 other complex isolates had a guanine residue. PCR-restriction fragment length polymorphism analysis of oxyR with restriction endonuclease AluI in an additional 255 complex isolates from widespread intercontinental sources confirmed and extended the unique association of adenine at position 285 with M. bovis isolates.