The Yarlung Zangbo River is the largest river on the Tibetan Plateau, which is considered a hotspot of biodiversity conservation efforts. In this study, the fish fauna of the middle and lower Yarlung Zangbo River was investigated, to explore the species composition and spatial patterns of fish assemblages as well as the associated environmental factors. Thirty-four sites across an elevation of 589 to 3410 m were sampled in 2015 and 2016. A total of 35 species—28 native and seven exotic—was collected. Of these species, four were described for the first time, 14 were endemic to the Yarlung Zangbo-Brahmaputra River drainage and five are considered endangered. Cluster analysis revealed significant spatial changes in fish assemblages along the longitudinal gradient. Two site-groups separated by the Yarlung Zangbo Grand Canyon were identified, corresponding to midstream and downstream. In the midstream, fish assemblages were characterized by typical plateau fish species (e.g., Schizopygopsis younghusbandi, Schizothorax oconnori, Triplophysa stenura, Triplophysa brevicauda, Schizothorax macropogon, Triplophysa tibetana and Ptychobarbus dipogon), while in the downstream fish assemblages were characterized jointly by plateau species (e.g., Schizothorax curilabiatus and Schizothorax molesworthi) and oriental species (e.g., Garra tibetana, Psilorhynchus homaloptera, Pseudecheneis sulcata, Exostoma tenuicaudata, Parachiloglanis hodgarti, Neolissochilus hexagonolepis and Garra yajiangensis). Canonical correspondence analysis (CCA) showed that altitude, channel width and the density of macroinvertebrates were the main environmental factors determining the spatial distribution of fish assemblages. This study is an important step towards understanding fish assemblages in the middle and lower Yarlung Zangbo River and will help with future efforts to protect aquatic communities and their habitats.