The present study was undertaken at the Department of Veterinary Public Health, College of Veterinary Science and Animal Husbandry, Anand to study prevalence of Mycobacterium tuberculosis and Mycobacterium bovis infections in human and animal population through tuberculin testing in cattle (single intra-dermal testing) and in human (Mantoux test) and by isolating and characterizing Mycobacteria from human, animal and environment, to elucidate etiologic significance of Mycobacteria, and to differentiate Mycobacterium tuberculosis and Mycobacterium bovis by biochemical tests and polymerase chain reaction.All the isolates of Mycobacteria were identified and differentiated by PCR by using specific primers for the detection of p34 gene that codes for 34-kDa for the detection of Mycobacterium tuberculosis complex. Mycobacterium bovis and M. tuberculosis were differentiated by using specific primer for hupB gene that codes for histone like protein. All isolates of Mycobacterium tuberculosis and Mycobacterium bovis yielded desired amplification of 336 and 309 bp, respectively.