Ixodid ticks transmit a variety of pathogens by blood feeding. Here, we report computational identification of two multigene families of defensin-like peptides (DLPs) in the Ixodes scapularis genome, one corresponding to scapularisin and the other named scasin. Members in the scapularisin family share high sequence similarity to some antibacterial ancient invertebrate-type defensins (AITDs) isolated from primitive insects, arachnids, bivalvia, and fungi whereas scasins represent a novel family of DLPs identified by their overall acidic molecular surface and low sequence similarity to any known defensins. Codon-substitution models support neutral evolution in scapularisins but strong positive selection signal was found throughout the molecules of scasins. The synthetic γ-core region of scapularisin-20 exhibits a wide-spectrum of antimicrobial activity at micromolar concentrations. The finding of extensive gene expansion of DLPs in a vector arachnida may be valuable in the understanding of its role in pathogen transmission.