The present invention relates to the discovery of a DNA sequence in sheep types of M. paratuberculosis that differs from the homologous sequence in cattle types of M. paratuberculosis. The invention also provides a nucleic acid amplification technique based on these differences that can be used to distinguish strains of the cattle type from strains of both the sheep types of M. paratuberculosis. The invention also relates to use of these sequences in a nucleic acid amplification technique to distinguish all strains of M. paratuberculosis from other strains of the MAI complex and from strains of the M. tuberculosis complex.