Abstract: Salmo trutta fario is a second-level protected wild animal in the Xizang Autonomous Region, distributed only in the Yadong River segment in Yadong County, Shigatse City, Xizang. In recent years, due to overfishing and changes in hydrological conditions, the population resources of wild S. trutta have rapidly declined. Due to the difficulty in obtaining samples, there are few studies on the feeding habits of S. trutta. The analysis of the food composition of S. trutta is helpful for understanding its population dynamics and ecological functions, and it is of great significance for the protection and restoration of fishery resources. In this study, 26 wild S. trutta were collected from Yadong River in Yadong County, and 22 intestinal contents were obtained. Two pairs of universal primers targeting the highly variable regions of 18S rRNA V4 and COI genes were used to identify the food composition of intestinal contents. The results showed that a total of 17 food taxa were obtained: Mantodea, Calanoida, Cladocera, Araneae, Coleoptera, Cyclopoida, Diptera, Ephemeroptera, Haplotaxida, Hemiptera, Hymenoptera, Lepidoptera, Lumbriculida, Melosirales, Parachela, Ploima, and Trichoptera. Among all food taxa, Diptera had the highest frequency of occurrence, percent of occurrence, weighted percent of occurrence, and relative read abundance (18S rRNA were 81.82%, 26.87%, 35.53%, and 53.77%; COIwere 81.82%, 29.03%, 39.09%, and 45.14%), followed by Lepidoptera (18S rRNA were 59.09%, 19.40%, 19.02%, and 14.33%; COIwere 45.45%, 16.13%, 16.32%, and 21.39%). Paired Wilcoxon test showed that the amplicon sequence variants (ASVs) of food taxa amplified by the 18S rRNA region were significantly higher than those amplified by the COI region, and species annotation chi-square test showed that the annotation success rate of the COI region was higher (χ²=43.47, df=4). Overall, S. trutta mainly feeds on aquatic insects, with Diptera being the main food composition. In addition, planktonic animals and planktonic plants were also found, which may be accidentally ingested during predation. In the process of analyzing food composition using DNA barcoding, there are differences in the results obtained with different molecular markers, and the results obtained by using two or more molecular markers together will be more comprehensive. The results of this study can provide reference for understanding the survival status of wild S. trutta populations and formulating protection policies.