Charophytes from the middle to late Eocene Kangtuo Formation in the Gaize Basin, central Tibet, are studied from the perspectives of taxonomy, palaeoecology, and biogeography. In the upper member of the Kangtuo Formation of the Mengdangle (MDL) section, southern Gaize Basin, two charophyte species were studied, including Hornichara jianglingensis comb. nov. and Chara cf. wujinensis comb.nov. The flora lives in the temporary pond's depositional environment, based on sedimentological analysis and fossil species occurrence. A palae-obiogeographic analysis of the charophyte assemblage suggests that H. jianglingensis was widely distributed in China basins during the middle to late Eocene, and later as a regional species in the coastal area in the Oligocene. H. jianglingensis and C. cf. wujinensis disappeared during the Oligocene in the interior of the Tibetan Plateau, and it was likely attributed to the rapid uplift of the Central Tibetan Valley.