We have used a Mycobacterium avium subsp. paratuberculosis (MAP), strain Linda, genomic library to select clones containing copies of IS900 and to sequence their flanking regions. This approach has determined sequences from 13 of the 16 loci predicted for this strain by RFLP analysis. 12 of these loci are located immediately upstream of ORFs inside a putative ribosome binding motif AGGAGA. Analysis of homologous genomic regions in Mycobacterium avium subsp. avium, indicates that IS900 insertion effects deletion of the terminal base to this motif. The relative orientation of IS900 with these ORFs is consistent with the transcription of hed. Only one locus contained an IS900 insertion inside an ORF the orientation of which was consistent with the transcription of p43. Using these data we have developed a rapid PCR multiplex typing method based upon the presence/absence of IS900 in these loci. This method uses an anchor primer designed from the 3' end of IS900 in conjunction with 13 locus specific primers. These are designed to give PCR products that differ by at least 50bp, visualised as a PCR product 'ladder' on 1.5% agarose electrophoresis gels. IS900 loci that are not filled or have undergone genomic re-arrangements relative to the Linda strain, do not produce PCR products. We have screened over 50 strains of MAP using this system, including representatives of all previously published RFLP types from environmental, sheep, cattle, goat, primate and human sources. Results show that the multiplex system is not as variable as RFLP. However consistent differences were seen between IS900 loci in sheep strains and MAP from other sources, probably due to genomic re-arrangements. Unique multiplex profiles were detected in some strains that had identical RFLP patterns. In addition, isolates from two human isolates and one primate isolate gave a unique multiplex profile.