Abstract: We analysed spatial pattern in the fish assemblage structure along a longitudinal gradient of the Upper Yangtze River and the Three Gorges Reservoir. We tested the hypothesis that shifts from lotic to lentic environment affect the richness and structure of the fish assemblage. Samplings were carried out from 2005 to 2012 in four zones:(1) Hejiang reach, river upstream from the reservoir; (2) Mudong reach, upper part of the reservoir; (3) Wanzhou reach, middle part of the reservoir, and (4) Zigui reach, lower part of the reservoir. A total of 368706 fish representing 132 native species of 17 families were collected during the study period with Cyprinidae as the dominant group. The results showed that the native species richness decreased while the non-native species increased from river (Hejiang reach) to reservoir (Zigui reach). Patterns in fish assemblage ordination evaluated by correspondence analysis reflected a clear division of the riverine and reservoir zones. Uppermost sampling stations were characterized by species characteristic of flowing waters, whereas in the lowland most lentic species were captured. Further, 22 species, three functional groups (rheophilic, equilibrium, insectivorous) were identified for the upper reach and 16 species, three functional groups (herbivorous, planktivorous, stagnophilic) for the lowland reach by indicator species analysis (P<0.05). Therefore, it is evident that impoundment of the Three Gorges Reservoir is the major driving factor to structuring the fish assemblage structure along the longitudinal gradient from river to the reservoir. Different fisheries management actions should be made to conserve or rehabilitate native fish assemblages and control invasive non-native species.