Tuberculosis (TB) in domestic and wild ruminants is mostly caused by Mycobacterium bovis (MB). However, the present paper describes the first report of TB of antelopes (Antelope cervicapra) and chinkara (Gazella bennettii) due to Mycobacterium tuberculosis (MT) in India. These wild hosts may represent a vehicle for the dissemination of MT infection to domestic livestock or human. MT was isolated by culture employing the MGITTM BACTECTM 960 (Becton Dickinson, BD) system and Middlebrook 7H10 Agar enriched with oleic albumin dextrose catalase (OADC) and Lowenstein-Jensen (LJ) medium supplemented with glycerol, but not sodium pyruvate. The isolates were confirmed as a member of the Mycobacterium tuberculosis complex (MTC) by using a commercial nested PCR that targeted the IS6110 sequence. Further confirmation of the isolates as MT strains was achieved by employing commercial line probe assay genotyping kits (Hain Lifescience, Germany) that specifically identifies MT within the MTC group.