Comparative genomic analyses have yielded valuable insights into conserved and divergent aspects of gene function, regulation, and evolution. Herein, we describe the characterization of a mouse beta-defensin gene cluster locus on chromosome 2F6. In addition, we present the evolutionary analysis of this cluster and its human, rhesus, and rat orthologs. Expression analysis in mouse revealed the occurrence of defensin cluster transcripts in multiple tissues, with the highest abundance in the urogenital tract. Molecular evolutionary analysis suggests that this cluster originated by a series of duplication events, and by positive selection occurring even after the rodent-primate split. In addition, the constraints analysis showed higher positive selection in rodents than in primates, especially distal to the six-cysteine array. Positive selection in the evolution of these defensins may relate not only to the evolving enhancement of ancestral host defense but also to functional innovations in reproduction. The multiplicity of defensins and their preferential overexpression in the urogenital tract indicate that defensins function in the protection and maintenance of fertility.