Kallikrein-related peptidase 4 (KLK4) is a secreted serine protease that degrades residual enamel proteins to facilitate their removal by ameloblasts, which increases mineralization and hardens the enamel. Mutations in human KLK4 cause hypomaturation amelogenesis imperfecta. Enamel formed by Klk4 null mice is normal in thickness and prism structure, but the enamel layer retains proteins, is hypomineralized, and undergoes rapid attrition following tooth eruption. We searched multiple databases, retrieved Klk4 and Klk5 from various mammalian genomes, and identified Klk4 in 47 boreoeutherian genomes. In non-Boreoeutheria, Klk4 was detected in only one afrotherian genome (as a pseudogene), and not in the other six afrotherian, two xenarthran, or three marsupial genomes. In contrast, Klk5 was detected in both marsupial and eutherian mammals. Our phylogenetic and mutation rate analyses support the hypothesis that Klk4 arose from Klk5 by gene duplication near the divergence of Afrotheria, Xenarthra and Boreoeutheria, and that functionally- differentiated Klk4 survived only in Boreoeutheria. Afrotherian mammals share the feature of delayed dental eruption relative to boreoeutherian mammals. KLK4 shortens the time required for enamel maturation and could have alleviated negative selection following mutations that resulted in thicker enamel or earlier tooth eruption, without reducing enamel hardness or causing dental attrition.