Strains of Mycobacterium avium subspecies paratuberculosis have been subdivided into three groups, Type I, II and III, by pulsed-field gel electrophoresis (PFGE). Type I strains comprise pigmented, slow growing strains that appear to have a host preference for sheep; Type II strains comprise non-pigmented strains with a very broad host range; Type III strains comprise non-pigmented strains that have PFGE profiles intermediate between Type I and II and exhibit biological characteristics from both groups. It is proposed that Type I strains are an evolutionary intermediate between Mycobacterium avium subsp. avium and Type III strains. We have used 2-D gel electrophoresis to characterize the proteomes of the three strain types to investigate the molecular basis for the biological differences observed. Type I and II strains were found to express Type-specific proteins. No Type-specific strains were identified for Type III strains. Type-specific proteins were identified by Matrix Assisted Laser Desorption Ionisation Mass Spectometry (MALDI MS). The relevance of these proteins to phenotype and evolutionary perspectives will be discussed.