Dams have well-documented ecological impacts on downstream river segments; however, long-term impacts of river impoundment have rarely been investigated in upstream reaches. Using data from long-term standardized surveys, we analyzed temporal changes in fish assemblages in the Yangtze River upstream of the Three Gorges Dam (TGD) before, during and after its construction. Our analysis indicated fish assemblage regime shifts in the two closer reaches in 2008, in accordance with the filling to 172.5 m in 2008; and in the other reach, farthest from the TGD, in 2011, indicating timing of the effects being related to distance. These shifts were evident in relative abundance of native fish species rather than non-native species and have altered community structures and functional groups. Relative abundance of the lotic guilds declined in the two closer reaches, but increased in the farthest. Invertivores declined, but piscivores and opportunistic life-history strategists increased in all reaches. We conclude that construction of TGD had led to significant changes in species distributions influenced by species functional traits. Our findings emphasize the need for long-term monitoring of fish assemblages before and after dam construction in order to understand ecological responses to hydrological changes for effective resource management in regulated rivers.