Triplophysa ferganaensis sp. nov. is described from the Shakhimardan stream, a small tributary of the Syr Darya, which does not reach the river in Fergana Valley. It can be distinguished from other valid Triplophysa loaches based on the following combination of characters: body smooth and scaleless, lateral line complete, posterior chamber of air bladder degenerated, inner gill rakers 10–11 on the first‐gill arch, outer gill rakers absent, vertebrae 4 + 35–36, 8 + 8 branched caudal‐fin rays, caudal peduncle depth 2.1–2.7 times its length, two supratemporal pores, dorsal‐fin origin closer to the caudal‐fin base than to the snout tip, caudal fin emarginated and pelvic‐fin tip reaching the anus. The new species can also be distinguished from its congeners based on the molecular analyses of mitochondrial cytochrome oxidase subunit I (coI) gene sequences. The phylogenetic position of this new species indicates that it is a sister taxon of Triplophysa tenuis.