Previous studies using IS900 RFLP and MPIL typing of Mycobacterium avium subspecies paratuberculosis (MAP) strains have shown distinct genomic differences between strains isolated from bovine and ovine origin suggesting that the clonal expansion of MAP in different species has led to the development of strain specificity for particular hosts. Seven strains of MAP isolated from 7 patients with Crohns disease (CD) were obtained from Dr S. Naser, University of Central Florida, USA. These were sub-cultured and checked for purity in MGIT (Becton Dickinson) liquid medium. 2 strains were isolated from patient breast milk and 5 from resected gut tissue. Growth was relatively rapid in 6/7 strains with good yields obtained in under 4 weeks. ZN staining of these cultures showed these strains all exhibited both ZN positive and ZN negative forms. The identity of each strain was confirmed as MAP by using PCR reactions specific for the MAP genome (gsd-mpa junction and MAP MIRU2 & 3 locus PCR). MPIL typing of each strain using PCR reactions specific for 14 different IS900 loci was performed to determine the number of loci filled with IS900 and any genomic re-arrangements associated with IS900. PCR products from IS900 Locus 5 were sequenced to determine SNP differences between strains. Results showed that all but one of the strains were MPIL type 3 (RFLP type B/C5) and were missing an IS900 insertion at Locus 5 resulting in the desA1 gene remaining intact. Strains could be further differentiated from bovine/ovine strains and a previous MPIL type 3 MAP strain BEN isolated from a human with CD by SNPs present in locus 5. These results suggest that the loss of IS900 from locus 5 in MAP and the presumed resumption of desA1 gene expression is associated with MAP strains isolated from within the inflamed gut wall of patients with CD.