The Three Gorges Dam on the Yangtze River is one of the largest dams in the world. The water level of the Three Gorges Reservoir (TGR) reached up to 135 m during the first stage of impoundment in 2003 and up to 156 m during the second stage of impoundment in 2006. The third stage of impoundment was conducted in September 2010, during which the water level in the TGR raised to 175 m. Previous studies showed that environmental changes that resulted from the two previous impoundments of the TGR have consequently caused changes in the species composition and structure of the fish communities in the TGR. However, only a few studies were conducted to determine the effect of the 175-m-deep experimental impoundment, which is the highest water level of the TGR, on the fish communities. In this study, we conducted surveys of the commercial catch over 167 days from 2010 to 2012 in five sections of the main stream (Zigui, Wushan, Yunyang, Fuling, and Jiangjin) located in the TGR and the upper Yangtze to reveal the spatiotemporal changes in the structure of the fish communities. A total of 87 fish species belonging to eight orders, 18 families, and 63 genera were recorded during these surveys. The number of species gradually increased from the front reservoir to the tail reservoir in the annual surveys. Cluster analysis (CA) and nonmetric multidimensional scaling (NMDS) resolved two groups of fish species at the five surveyed sections with Jaccard similarity of 58.60% (R=0.883, P<0.01). In particular, the species inhabiting the front and middle reservoirs, including the Zigui, Wushan, and Yunyang sections, were classified into one group. that had significant variations were observed among all years of the three sections. The species inhabiting the tail reservoir and the upper Yangtze River, including the Fuling and Jiangjin sections, were classified as another group that did not exhibit significant annual variations in the species compositions. Moreover, CA and NMDS showed that the spatial pattern of fish communities varied yearly. The annual increase in the similarity of fish communities was observed between the Yunyang and Fuling sections, and annual decrease was observed between the Yuyang and Zigui/Fuling sections as well as between the Fuling and Jiangjin sections. In summary, the front reservoir was dominated by lentic species, such as Hypophthalmichthys molitrix (Valenceinnes in Cuvier et Valenciennes),Pseudobrama simoni (Bleeker),Aristichthys nobilis (Richardson), and Hemiculter leucisculus (Basilewsky). The tail reservoir and upper Yangtze River were dominated by lotic species, such as Coreius heterokon (Bleeker),Coreius guichenoti (Sauyage et Dabry),Rhinogobio ventralis (Savage et Dabry), and Rhinogobio cylindricus (Günther). Additionally, species in the middle and tail reservoirs shifted from lotic species to lentic species with the increase in water level of the TGR. This study shows that the 175-m-deep experimental impoundment of the TGR immediately affected the structures of fish communities in the TGR, especially in the middle and the tail reservoirs, possibly because of the alteration of flow regimes that results from the increasing water level.