The changes in fish resources after the impoundment of the Three Gorges Reservoir have attracted considerable attention. To explore the fish assemblage structure and population dynamics of the major fish populations in the Three Gorges Reservoir, we sampled fish from the lower to upper sections of the reservoir from 2015 to 2016, and analyzed the age structures of 12 primary fish populations. A total of 89 fish species were identified, with 62, 62, and 85 species recorded in the lower, middle, and upper sections, respectively. Fish assemblages were dominated by Cyprinus carpio, Hemiculter spp., and Hypophthalmichthys molitrix in the lower section; Leiocassis longirostris, Pelteobagrus spp., Saurogobio spp., and Squalidus spp. in the middle section; and Rhinogobio spp. and Coreius spp. in the upper section. In contrast, fish assemblages exhibited spatial gradients from the perspective of settlement type, habitat use, and feeding habit. These populations were composed of ages 1–7 years, and 1–3 years individuals that dominated the populations with an average proportion of 88.51%. Our results revealed that the fish community showed spatially gradient changes, and the key fish populations tended to be of younger age. This study raises suggestions for the evaluation of the ecological effects of fishing ban and further fish resource management.