The ß-defensin is innate immune peptides with evolutionary conservation across a wide range of species. Here we characterized 17 unreported porcine β-defensin (pBD) genes using bioinformatic analyses. BLAST analysis was performed against the current pig genome assembly to identify β-defensin related sequences in the pig genome using previously reported β-defensin sequences including pigs, humans and cattle. We identified sequences containing the 6-cystein motif which is the characteristics of β-defensins in their mature peptide region, and identified 27 β-defensins in which 10 were previously reported. Comparative and phylogenetic analyses of pDB genes together with those of humans and cattle were performed, showing that the syntenic conservation of β-defensin genes among these species. Our gene expression analysis using 17 pBD genes across 15 tissues from a 2-week-old NIH miniature pig using semi-quantitative RT-PCR showed higher expression of pBD genes in tissues including small intestine, tongue, spleen kidney, liver, testis and thymus. Our results will be helpful to understand the functional differences or specificity among different pBDs.