Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne’s disease in cattle, sheep and goats. There are many methods for diagnosis of JD and among these, culture and identification is the “gold standard” for diagnosis of paratuberculosis in spite of its long turn over time. Works on mycobacterial genomics revealed that genomic reduction through the loss of large sequence polymorphisms is the major contributor of genomic diversity in M avium subspecies. LSPP 12 is present in mbt A gene of mycobactin synthesis operon replacing LSP 4 in M avium and LSPP12 internal sequence PCR can be used for specific detection of MAP. LSPP12 faecal PCR gave similar results as that of IS 900 PCR and only MAP DNA gave specific amplification with LSPP 12 PCR compared to mother mycobacteria used in this study.