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Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that play an essential role in innate immunity across taxa from invertebrates to vertebrates. The evolution system of AMP system has not been well explained in the literature. In this study, we cloned and sequenced AMP transcriptomes of three frog species, namely Rana dybowskii, Rana amurensis, and Pelophylax nigromaculatus, which are partially sympatric in northeast Asia, but show different habitat preferences. We found that each species contained 7 to 14 families of AMPs and the diversity was higher in species with a large geographic range and greater habitat variation. All AMPs are phylogenetically related but not associated with the speciation process. Most AMP genes were under negative selection. We propose that the diversification and addition of novel functions and improvement of antimicrobial efficiency are facilitated by the expansion of family members and numbers. We also documented significant negative correlation of net charges and numbers of amino acid residues between the propiece and mature peptide segments. This supports the Net Charge Balance Hypothesis. We propose the Cut Point Sliding Hypothesis as a novel diversification mechanism to explain the correlation in lengths of the two segments. [ABSTRACT FROM AUTHOR]