Vertebrate β-defensins comprise an important family of antimicrobial peptides that protect organisms from a diverse spectrum of bacteria, viruses, fungi, and protozoan parasites. Previous studies have shown a marked variation in the number of β-defensins among species, but the underlying reason is unclear. To address this question, we performed comprehensive computational searches to study the intact β-defensin genes from 29 vertebrates. Phylogenetic analysis of the β-defensin genes in vertebrates identified frequent changes in the number of β-defensin genes and multiple species-specific gene gains and losses that have been occurring throughout the evolution of vertebrates. The number of intact β-defensin genes varied from 1 in the western clawed frog to 20 in cattle, with numerous expansions and contractions of the gene family throughout vertebrates, especially among tetrapods. The β-defensin gene number in a species is relevant to the ever-changing microbial challenges from the environment that they inhabit. Selection pressure analysis shows there exist three amino acid sites under significant positive selection. Protein structural characteristics analysis suggests that structural diversity determines the diverse functions of β-defensins. Our study provides a new perspective on the relationships among vertebrate β-defensin gene repertoires and different survival circumstances, which helps explain how β-defensins have evolved.