Gymnocypris waddelli, a highland cold-water fish, is distributed among rivers and lakes of the southern Qinghai-Tibet Plateau. Although previously described as G. waddelli, specimens from the Pengqu River are morphologically distinguishable from those from Lake Yamzhog Yumco, which is the type locality of G. waddelli. We investigated morphological variations of G. waddelli specimens from Pengqu River and Lake Yamzhog Yumco using not only traditional morphological methods but also newly-developed geometric morphometric analysis; furthermore, mitochondrial Cytochrome b (Cyt b) gene analysis was employed to explore the phylogenetic position of the Pengqu River population. Our morphological analyses suggested that G. waddelli from the Pengqu River had an obtuse snout, nearly straight oral fissure, shorter lower jaw than upper jaw, broad mouth and sparse inside gill rakers of the first arch relative to the Lake Yamzhog Yumco population. The strongly supported monophyly of Pengqu population according to mitochondrial datasets was obtained, indicating genetic differences between Pengqu River and Lake Yamzhog Yumco populations. Given their substantial genetic and morphological divergence, specimens from Pengqu River warrant recognition as a distinct species, here described and named Gymnocypris pengquensis.