Solute carrier 12 (Slc12) is a family of electroneutral cation-coupled chloride (Cl−) cotransporters. Na+/K+/2Cl− (Nkcc) and Na+/Cl− cotransporters (Ncc) belong to the Nkcc/Ncc subfamily. Human and mouse possess one gene for the Na+/Cl− cotransporter (ncc gene: slc12a3), whereas teleost fishes possess multiple ncc genes, slc12a3 (ncc1) and slc12a10 (ncc2), in addition to their species-specific paralogs. Amphibians and squamates have two ncc genes: slc12a3 (ncc1) and ncc3. However, the evolutionary relationship between slc12a10 and ncc3 remains unresolved, and the presence of slc12a10 (ncc2) in mammals has not been clarified. Synteny and phylogenetic analyses of vertebrate genome databases showed that ncc3 is the ortholog of slc12a10, and slc12a10 is present in most ray-finned fishes, coelacanths, amphibians, reptiles, and a few mammals (e.g., platypus and horse) but pseudogenized or deleted in birds, most mammals, and some ray-finned fishes (pufferfishes). This shows that slc12a10 is widely present among bony vertebrates and pseudogenized or deleted independently in multiple lineages. Notably, as compared with some fish that show varied slc12a10 tissue expression profile, spotted gar, African clawed frog, red-eared slider turtle, and horse express slc12a10 in the ovaries or premature gonads. In horse tissues, an unexpectedly large number of splicing variants for Slc12a10 have been cloned, many of which encode truncated forms of Slc12a10, suggesting that the functional constraints of horse slc12a10 are weakened, which may be in the process of becoming a pseudogene. Our results elaborate on the evolution of Nkcc/Ncc subfamily of Slc12 in vertebrates. [ABSTRACT FROM AUTHOR]