Abstract: The Lhasa River is the biggest tributary of the Yarlung Zangbo River, Tibet, China. Non-native fishes were found recently in the basin, but the distribution of non-native fishes and fish assemblage in the whole basin were poorly known. We examined fish assemblage and its relationship with the environmental factors in the basin in 2010, 2012 and 2014. 25 fish species were collected, and 13 of them were non-native fishes. Non-native fishes were distributed at 19 of 42 sampling sites, including 12 wetland sites, 6 river sites and 1 reservoirs above dam. Most non-native fishes occurred in the downstream with the exception of Carassius auratus in the upstream and Cyprinus carpio, Aristichthys nobilis as well as Misgurnus anguillicaudatus in the middlestream of the river. The most widely distributed and abundant native fishes were Schizopygopsis younghusbandi younghusbandi, Triplophysa orientalis and T. tibetana, and the most widely distributed and abundant non-native fishes were M. anguillicaudatus, C. auratus and Pseudorasbora parva. Canonical correspondence analysis indicated that the fish assemblage structure was impacted by both local environmental and regional environment factors, and the non-native fishes and native fishes had different relationships with the environment factors (elevation and substrate).