Abstract: This study aimed to analyse the temporal and spatial variations in the trophic structures of fish assemblages in the Chishui River, the last undammed tributary of the upper Yangtze River. Fish samples were collected from different sections (the headwater, the upstream, the midstream and the downstream) along the longitudinal gradient during the flood season (June) and dry season (December) of 2023. Stable carbon and nitrogen isotope values (δ¹³C and δ¹⁵N) were measured for 2016 fish specimens, belonging to 3 orders, 13 families and 83species. Temporal and spatial variations were analyzed by using MixSIAR and SIBER. Results showed that the δ¹³C values of fish species ranged from -29.36‰ to -15.81‰, while δ¹⁵N values ranged from 6.27‰ to 16.66‰. The δ¹³C and δ¹⁵N values in the upstream and midstream were significantly higher in the flood season than that in the dry season (P<0.05), while those in the downstream showed non-significant seasonal differences. Along the longitudinal gradient, the δ¹³C and δ¹⁵N values increased gradually first and then decreased in the downstream, with the maximum values occurring in the midstream. Trophic levels of fish assemblages ranged from 1.21 to 4.46, with average trophic levels significantly higher in the flood season than those in the dry season (P<0.05). Fish assemblages in the midstream exhibited significantly higher average trophic levels than those in other sections (P<0.05). The analysis of trophic structure indicators revealed significant temporal and spatial differences in fish assemblages. Temporally, for most sections (except for the downstream), higher Nitrogen Range (NR), Total Area (TA), Mean Nearest Neighbor Distance (MNND), and Standard Deviation of Nearest Neighbor Distance (SDNND) were observed in the flood season, suggesting greater trophic niche differentiation and more complex food webs than that in the dry season. Spatially, NR, Carbon Range (CR), and TA increased gradually along the longitudinal gradient, suggesting greater trophic diversity and increasingly complex food web downstream. Significant differences were observed among different feeding guilds, with the piscivore fishes exhibited the highest trophic level. These findings provide critical insights into the ecosystem structure and function of the Chishui River and offer scientific guidance for fish biodiversity conservation and ecological restoration efforts.