Studies of alpha-2-macroglobulin (α2M), a universal protease inhibitor, have indicated that it plays a unique and critical role in the innate immune system of vertebrate and invertebrate animals. The distinctive mechanism of pathogen inhibition – through physical entrapment of the pathogen-derived protease – makes α2M an ideal candidate for molecular evolutionary analysis. Furthermore, recent studies revealed that the Osteichthyes are characterized by levels of α2M diversification that exceed those recorded in other animal groups. Our study of Danio rerio (zebrafish) indicated that (1) two distinct lineages of α2M and α2M-like isoforms exist and (2) at least some codons in the functional domains of α2M have been subjected to positive Darwinian selection. The findings of several hot-spots for nonsynonymous substitutions in the two functional domains such as bait region and receptor binding domain, suggest that host-immune selection have played a dominant role in these two genomic regions of α2M. The presence of two, non-monophyletic α2M lineages in zebrafish provides compelling evidence of an ancient gene duplication event. The accelerated rate of nucleotide substitution in the functional domains of α2M is consistent with similar observations of other immune system components.