It is vital to study the spatial distribution characteristics and formation mechanism of biodiversity to enable its conservation. However, how spatial changes affect fish community structure is controversial. The Yarlung Zangbo River is the highest river in the world and nurtures the most distinctive plateau ecosystem and fish resources worldwide. The Yarlung Zangbo River Grand Canyon is located in the lower reaches of the Yarlung Zangbo River and is the deepest canyon in the world. Due to the complex topography and inconvenient transportation in the Yarlung Zangbo Grand Canyon, research on fish community structure and diversity is also unknown in the Motuo reach. This investigation presents the first analysis of the spatial variation in structure and diversity of fish communities in the Motuo reach of the Yarlung Zangbo Grand Canyon. Based on fishery resource survey data in the Motuo reach and Buqun Lake in spring (April) of 2015, and spring (March to April) and autumn (November) of 2017. Spatial variation in the structure and diversity of fish communities in the Motuo reach was determined. The results indicated that 18 fish species were caught, which belonged to 2 orders, 4 families, and 13 genera. There were 2 non-native fish species, including Cyprinus carpio and Pseudorasbora parva. The composition of species has changed compared with historical data. On one hand, there were some differences in the results caused by different classification and identification methods. Historically, morphological identification was primarily used, but molecular genetic methods were used in this study. On the other hand, the fishing methods and intensity were different. Electric capture was used for the first time in this survey. We found three new fish species by morphological and molecular genetics. These new species belonged to Schizothorax, Garra, and Exostoma, and were tentatively named Schizothorax sp., Garra sp., and Exostoma sp., respectively. There was significant spatial variation in species composition between the main stream and its tributaries; the main stream was primarily composed of large- and medium-sized fish, whereas the tributaries consisted primarily of small fish. The predominant species in the fish communities were also different between the main stream and its tributaries; however, Schizothorax curvilabiatus was the dominant species in all water bodies. First, the main factors affecting the composition of the river fish community were the spatial changes in the physical and chemical environment. Second, the variability of environmental factors led to the spatial heterogeneity of fish community structure. Third, the spatial differences in fish composition were also related to their living habits and ecological needs. S. curvilabiatus had the highest relative biomass and abundance. The mean relative resource was 9844.7 g/h by electric fishing, 446 g per gill net in the main stream, and 486.4 g/h by electric fishing in tributaries. The range of Shannon-Wiener diversity index H′ varied from 0.95 to 1.77, and the range of the Margalef species richness index D? was from 0.58 to 1.13, with the Pielou evenness index J? being from 0.37 to 0.77. There was a difference between the main stream and its tributaries according to each diversity index, and all diversity indexes in the main stream were lower than the corresponding indexes in tributaries. The unsuitability of the abiotic environment and the low availability of ecological niches and habitat may be the reasons for the low diversity in this reach and may also have contributed to the difference in the degree to which natural disasters threaten tributaries and main streams and the abundance of the organisms in the feed. This survey aims to provide background information to reveal the evolution of fish resources in the lower reaches of the Yarlung Zangbo River under human disturbance after hydropower development and provide a scientific basis for the protection of fish species diversity and fishery resource management in the Yarlung Zangbo Grand Canyon National Nature Reserve.