Crustins are cysteine-rich cationic antimicrobial peptides (AMPs) found in decapod crustaceans. Six novel crustin genes (Paj-CrusIc, Id, Ie, If, IIb and IIc) were identified in the morotoge shrimp, Pandalopsis japonica. Deduced amino acid sequences of isolated Paj-Crus genes ranged from 99 to 178 amino acid residues (10.6–17.8 kDa). Sequence analysis of nine isolated Paj-Crus genes and 100 different crustins from various decapod crustaceans revealed that a splice site and KXXXCP motif within the WAP domain may be the main criteria for classifying type I and II crustins, suggesting that the two types of crustin genes may have been generated by different processes. We also identified three intron-less crustin I genes (Paj-Crus Id, Ie and If) for the first time, which may have been generated by gene duplication. The tissue distribution profiles showed that Paj-CrusI genes were expressed predominantly in the gill and epidermis, whereas Paj-CrusII genes were expressed ubiquitously, suggesting that the two types of crustins may play different roles in various tissues or under different physiological conditions. Differing from previous results, hemocyte-specific crustin was not isolated from Pandalopsis japonica. This study showed that both types of crustin genes (types I and II) exist in decapod crustaceans and their primary structure and expression profiles differ from each other, suggesting that they may play different biological roles. This will help to extend our knowledge of the crustacean innate immune response, which will provide important basic information of shrimp immunity against various pathogens.