Neurotensin (NTS) plays important roles in neurotransmission and neuromodulation in the nervous system. NTS exerts its effects mainly by binding to the neurotensin receptor 1 (NTSR1) and receptor 2 (NTSR2) that belong to the G protein-coupled receptor superfamily. While studies on NTS and NTSR have been conducted mainly in mammalian systems, little is known about this ligand-receptor pair in nonmammalian species. Using a basic local alignment search tool combined with our previous identification of bullfrog Lithobates catesbeianus NTSR1 and NTSR2, we can define the evolutionary lineage of NTS and NTSR in vertebrates. Fish may have only one NTSR, which is orthologous to amphibian and mammalian NTSR1. Amphibian and mammalian species have two lineages of NTSR1 and NTSR2 subfamilies. While amphibian and mammalian NTSRs have overall structural similarity within the given subfamilies, they exhibit different pharmacological features and signal transduction pathways. This review will discuss the phylogenetic history of the G protein-coupled NTSRs, the structural features that may influence their pharmacological properties and signal transduction mechanisms, and the molecular interactions between NTSR1 and NTSR2 in vertebrates.