We have recently demonstrated that placental mammalian species such as pig and dog express a novel proapoptotic protease, caspase-15, whereas mouse and humans lack this enzyme. Here we investigated the evolutionary fate of the caspase-15 gene in different mammalian lineages by analyzing whole-genome shotgun sequences of 30 mammalian species for the presence of caspase-15 orthologs. Caspase-15 gene sequences were found in representatives of all major mammalian clades except for the superorders Afrotheria (tenrec, rock hyrax, and elephant) and Euarchontoglires (rodents, rabbit, tree shrew, and primates), which either lacked any caspase-15-like sequences or contained mutated remnants of the caspase-15 gene. Polymerase chain reaction screenings confirmed the results of the database searches and showed that the caspase-15 gene is expressed not only in various placental mammals but also in the marsupial, Monodelphis domestica. The observed species distribution implies that caspase-15 has originated in an early ancestor of modern mammals and has been conserved, over more than 180 Myr, in marsupials and many placental mammals, whereas it was independently lost in 2 phylogenetically distant clades of placental mammals, that is, Afrotheria and Euarchontoglires. Our data suggest that the inactivation of the caspase-15 gene was not counteracted by, and may even have been driven by, evolutionary constraints in these clades, and therefore, caution against the uncritical use of gene absence for the inference of phylogenetic relationships.