Important for calcium homeostasis, TRPV5 and TRPV6 are calcium-selective channels belonging to the transient receptor potential (TRP) gene family. In this study, we investigated the evolutionary history of these channels to add an evolutionary context to the already available physiological information. Phylogenetic analyses revealed that paralogs found in mammals, sauropsids, amphibians, and chondrichthyans, are the product of independent duplication events in the ancestor of each group. Within amniotes, we identified a traceable signature of three amino acids located at the amino-terminal intracellular region (HLH domain). The signature correlates well with both the duplication events and the phenotype of fast inactivation observed in mammalian TRPV6 channels. Electrophysiological recordings and mutagenesis suggest that calcium-induced fast inactivation represents an evolutionary innovation that emerged independently after gene duplication.