The ovine major histocompatibility complex (MHC) remains poorly characterized compared with those of other livestock species. Molecular genetic analysis of the bovine MHC has revealed considerable haplotype and allelic diversity that earlier serological analysis had not detected. To develop cellular and molecular tools to support development of vaccines against intracellular pathogens of sheep, we have undertaken a molecular genetic analysis of four distinct ovine MHC haplotypes carried by two heterozygous Blackface rams. We have identified 12 novel class I transcripts and used a class I sequence-specific genotyping system to assign each of these transcripts to individual haplotypes. Using a combination of phylogenetic analysis, haplotype and transcript expression data, we identified at least four distinct polymorphic class I MHC loci, three of which appear together in a number of combinations in individual haplotypes. The haplotypes were further characterized at the highly polymorphic Ovar-DRB1 locus, allowing selection of the progeny of the two founder rams for the establishment of an MHC-defined resource population.