Background: beta-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. beta-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the beta-defensin gene repertoire in sheep using the bovine genome as the primary reference.Results: All 57 currently predicted bovine beta-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (> 70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the beta-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 beta-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 beta-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential beta-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species.Conclusions: This is the first comprehensive analysis of beta-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of beta-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.