In Australia and other countries the distinction between ovine Johne's disease (OJD) and bovine Johne's disease (BJD) at the microbiological level plays an important role in the development and implementation of control and evaluation programmes for both forms of this insidious disease. However, the differences between the sheep and cattle strains of Mycobacterium avium subsp. paratuberculosis at the DNA level that result in the different host specificities are still extremely poorly understood. To date the DNA techniques that exist to differentiate these strains provide excellent tools for epidemiological investigations and diagnosis of Johne's disease but have yielded little if any insight between the relationship of genotype and phenotype. In this study an extensive comparison was made between the sheep and cattle strains of Mycobacterium avium subsp. paratuberculosis using a variety of modern DNA and protein based techniques including: representational difference analysis (RDA), polymerase chain reaction (PCR) and sequencing, microarray, two dimensional electrophoresis proteomics and surface enhanced laser desorption-ionisation (SELDI) proteomics. The majority of these techniques were used in conjunction with the recently completed Mycobacterium avium subsp. paratuberculosis K10 genome sequence in order to identify those genes or proteins found to have unique characteristics with either strain. Using these techniques a number of differences between the sheep and cattle strains of Mycobacterium avium subsp. paratuberculosis were identified. The results from this study are discussed.