Defensins are cysteine-rich cationic peptides that function in antimicrobial defense in both invertebrates and vertebrates. Three main groups of animal defensins are known: insect defensins; mammalian alpha-defensins and vertebrate beta-defensins. It has been difficult to determine whether these molecules are homologous or have independently evolved similar features, but overall the evidence favors a distant relationship. The best evidence of this relationship is structural, particularly from their overall three-dimensional structure and from the spacing of half-cystine residues involved in intra-chain disulfide bonds. Some evidence favors a closer relationship between vertebrate beta-defensins and insect defensins than between the two groups of vertebrate defensins. Examination of nucleotide substitutions between recently duplicated mammalian defensin genes shows that the rate of nonsynonymous (amino-acid-altering) substitution exceeds that of synonymous substitution in the region of the gene encoding the mature defensin. This highly unusual pattern of nucleotide substitution is evidence that natural selection has acted to diversify defensins at the amino acid level. The resulting rapid evolution explains why it is difficult to reconstruct the evolutionary history of these molecules.