-defensins are small cationic proteins with potent immunoregulatory and antimicrobial activity. The number of genes encoding these peptides varies significantly between and within species but they have not been extensively characterised in the horse. Here, we describe a systematic search of the Equus caballus genome that identified a cluster of novel -defensin genes on Chromosome 22, which is homologous to a cluster on bovine Chromosome 13. Close genomic matches were found for orthologs of 13 of the bovine genes, which were named equine -defensins (eBD) 115, eBD116, eBD117, eBD119, eBD120, eBD122a, eBD123, eBD124, eBD125, eBD126, eBD127, eBD129 and eBD132. As expression of the homologous cluster in cattle was limited to the reproductive tract, tissue sections were obtained from the testis, caput, corpus and cauda epididymis and the vas deferens of three stallions and from the ovary, oviduct, uterine horn, uterus, cervix and vagina of three mares. Using a quantitative real-time polymerase chain reaction approach, each of the novel -defensin genes showed distinct region-specific patterns of expression. Preferential expression in the caput epididymis of these novel defensins in the stallion and in the oviduct in the mare suggests a possible role in immunoprotection of the equine reproductive tract or in fertility.