Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacterium avium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.