The Kizil River belongs to the Kashgar River system in the southern Xinjiang Uygur Autonomous Region. Inhabited by a variety of indigenous fishes, the food habits and trophic levels of the fish in the Kizil River have yet to be investigated. In this study, the food composition and trophic levels of five indigenous fish species from the Kizil River were determined. To this end, 46 samples of Schizothorax biddulphi, Schizothorax eurystomus, Schizothorax irregularis, Ttriplophysa yarkandensis and Triplophysa tenuis from the Kizil River were evaluated. The fish samples were analyzed for the morphological traits of the five feeding organs using traditional morphological methods. Furthermore, their intestinal food composition was analyzed using high-throughput sequencing techniques, and carbon and nitrogen stable isotopes were measured to analyze their trophic levels. δ¹⁵N and δ¹³C of Schizothorax and Triplophysa were used to construct the trophic structure and calculate the values of ecological indicators and parameters of the biotope. The results showed that the morphological differences of the feeding organs of the five indigenous fish species were comprised mainly of the intestinal length ratio and lip thickness. The two highland loaches had the same feeding organ morphology, a short intestinal length ratio, a small mouth cleft, and a slender body. A total of 4442842 valid sequence fragments were obtained from 46 samples using Illumina high-throughput sequencing. The lowest number of sequences in the samples was 60269, while the highest was 134,129, and the average number of sequences was 96583±16515. A total of 554 OTUs were obtained from 46 samples after clustering. The main bait organisms found in the stomach of the 46 specimens belonged to Chordata, Streptophyta, and Nematoda, among which the gut length ratio of S. irregularis was about 2.95. The main food source was primary producers, including Tamarix hispida, Glycine soja, and Nitraria tangutorum. During foraging in the riverbed substrate, the small individual benthic nematodes of S. irregularis were accidentally ingested by S. irregularis, resulting in a higher abundance of nematodes in its intestine. S. eurystomus had a shorter intestinal length ratio of approximately 2.56, with the main food source being primary producers, such as Brachypodium distachyon, which also prey on some small fish. S. eurystomus scrapes attached plant debris by means of sharp cuticles and wider mouth fissures. Benthic nematodes passively enter the gut of the wide-mouthed schizothorax when feeding. S. biddulphi has a relatively short gut length, with a ratio of approximately 2.38, and feeds mainly on Schizothorax fish, followed by Citrus latifolia, S. eurystomus and S. biddulphi were dissected, and a large number of small Schizothorax fish were found in their intestines. The intestinal length ratio in T. yarkandensis and T. tenuis were both less than one, and the proportion of Schizothorax fish in the intestine was over 95% of the food contents. Large plants and sand particles were also found in the highland loach, most likely ingested while chasing prey, due to water disturbance, via accidental ingestion. The intestinal contents of the two types of highland loach and S. biddulphi were similar while those of S. irregularis and S. eurystomus were similar. S. biddulphi, S. eurystomus, S. irregularis, T. yarkandensis, and T. tenuis δ¹⁵N ranged from 4.32‰ to 10.13‰, 1.94‰ to 9.78‰, 3.67‰ to 7.58‰, 2.22‰ to 11.78, and 4.61‰ to 11.88, respectively. Similarly, their δ¹³C ranged from ?27.36‰ to ?14.89‰, ?27.81‰ to ?13.87‰, ?26.09‰ to ?17.64‰, ?28.80‰ to ?15.47‰, and ?27.21‰ to ?18.79‰, respectively. The trophic levels of S. biddulphi, S. eurystomus, and S. irregularis were 3.19, 2.78, and 2.52, respectively, while those of T. yarkandensis and T. tenuis were 3.44 and 3.55, respectively. The average trophic diversity and feeding sources of Schizothorax were higher than those of Triplophysa, while the trophic level, density of species aggregation, and evenness of species aggregation of Triplophysa were higher than those of S. eurystomus. S. eurystomus is an omnivorous bi-phytophagous fish, whereas S. biddulphi is an omnivorous bi-carnivorous fish. T. yarkandensis and T. tenuis are both carnivorous fish and the top predators of the Kizil River, associated with material endpoints and energy transfer in the basin. The findings presented in this study provide insights into the food web of the Kizil River by exploring the relationships and trophic levels of fish in this habitat. In this study, the mean trophic levels of T. yarkandensis and T. tenuis, among others, are estimated to evaluate their ecological roles in the river ecosystem, improving our understanding of their feeding ecology and providing fundamental data for the conservation and rational use of fish resources in the Kizil River.