An isolate of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) was cultured from the distal ileum of a sheep that had been diagnosed with Johne's Disease. Comparative genomic hybridization and short sequence repeat typing were used to characterize this isolate as typical of North American Johne's Disease sheep isolates. Genomic DNA was isolated and used as a template for sequencing on the Roche Genome Sequencer 20 System. Pyrosequencing resulted in nearly 18-fold coverage of the genome and the resulting sequence fragments assembled into 550 contigs. Directed PCR followed by Sanger sequencing was subsequently used to close gaps in the assembly, resulting in fewer than 100 contigs. Analysis of the sequence identified several regions encoding genes that lack homology to other sequenced mycobacteria as well as regions with no homology to all publicly available sequences. Several regions contained homologues to sequences from Nocardia, Burkholderia, and Frankia isolates and encoded a variety of metabolic enzymes and transport proteins. Previously reported large sequence polymorphisms were identified in the genome sequence as well as a glycopeptidiolipid biosynthesis gene cluster. The draft and finished sheep isolate sequences will be made available to the research community. It is expected that the completed genome sequence will impart novel insights into the unique biology of M. paratuberculosis sheep isolates as well as provide a basis for further comparative genomic studies.