Mycobacterium avium supsp. avium (M. avium) and Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) share a genetic identity of more than 95% and presently, only a few M. paratuberculosis specific fragments have been described. However, the fundamental differences in habitat and virulence between M. avium and M. paratuberculosis seem to indicate, that factors exist, that condition these differences. It is possible, that identification of such genetic factors will offer an opportunity to improve existing diagnostic tests and perhaps allow new insight to the mechanisms of the disease Paratuberculosis. In this experiment the method of subtractive hybridisation of M. paratuberculosis against M. avium was used to isolate nine novel M. paratuberculosis specific sequence fragments of between 318 and 596 bp. Database search revealed little or no similarity with other mycobacteria, including M. avium. For each of the nine fragments specific primers were designed and assessment by PCR demonstrated, that the fragments, isolated by subtractive hybridisation, were present in the type strain (ATCC 19698) and a number of field strains of M. paratuberculosis but absent from M. avium and a number of mycobacterial species found in the environment. In Southern blots, all nine fragments appeared to represent probes capable of distinguishing between M. avium and nine other mycobacteria. RT-PCR with fragment specific primers resulted in amplified product of the expected size for all except one of the subtracted elements. This result seems to indicate, that eight of the nine fragments origin from areas of the genome that are expressed in vitro. The presence of open reading frames in these putative in vitro expressed M. paratuberculosis specific genetic elements implies, that some of these fragments origin from genes that encode M. paratuberculosis specific proteins and consequently will be of interest to isolate and characterise further - both with respect to possible antigens or virulence genes.