Ranatuerins are antimicrobial peptides of the innate immune system found in ranid frogs. We previously presented evidence that a positive selective sweep had fixed a single allele at the Ranatuerin2 locus in the northern leopard frog (Rana pipiens). In this paper, we further investigate the evolutionary history of ranatuerins as follows. First, we sequenced Ranatuerin2 in additional individuals of R. pipiens and related frog species and compared diversity and divergence at these sequences with that at four putatively neutrally evolving loci. Second, we asked whether the evolutionary patterns observed at Ranatuerin2 were typical for ranatuerin loci by sequencing our samples at a paralogous locus, Ranatuerin2b, and performing the same neutrality tests. Ranatuerin2b also showed strong and significant evidence of at least one selective sweep. Third, we used the neutral loci to independently resolve conflicting hypotheses about phylogenetic relationships among our study species. Both the neutral loci and the ranatuerin loci supported an older phylogeny inferred from allozyme data and strongly rejected a more recent phylogeny inferred from mitochondrial DNA. Finally, in order to test whether the sweep was driven by the evolution of substantially new peptide function, we used the phylogeny to reconstruct the hypothetical Ranatuerin2 peptide that existed before the sweep. We synthesized this peptide and tested its activity and that of the extant peptide against six bacterial pathogens of frogs. We observed antibacterial activity but found no significant functional differences between the two peptides.