Functional properties of the paracellular pathway depend critically on the set of claudins expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: HELIX syndrome caused by genetic variations in the CLDN10 gene, and Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis caused by genetic variations in the CLDN16 or the CLDN19 gene. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, 16 and 19 in both paraffin-embedded and frozen kidney sections from adult human, mouse and rat, using immunohistochemistry and immunofluorescence, respectively. Here CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junction in human and rodent kidney. A weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidney. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junction almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidney.