Abstract: Gut microbiota and host interactions co-evolve and develop into stably adapted microbial communities and play vital roles in maintaining the health of organisms. Diet is supposed to be an important driver of differences in gut microbiota, but previous studies would commonly use literature depictions, which are essential but inaccurate, to explain the effects of diet on the gut microbiota of wild birds. In this study, we collected intestinal samples from five sympatric flycatchers to compare the gut microbial differences using bacterial 16S rRNA genes from Illumina MiSeq platform. Over 1,642,482 quality-filtered sequences from 18 16S rRNA libraries were obtained and distinct compositions and diversities of gut microbiota were found in five flycatchers. Their gut microbiota is mainly from the four bacterial phyla of Proteobacteria, Firmicutes, Actinomycetes, and Bacteroidetes, but at the genus level showed a significant difference. Functional predictions revealed that the metabolic capacity of the gut microbiota of five flycatchers is greatly distinguished at KEGG level 3. And multiple food fragments showed a significant correlation with gut microbiota. Besides, the significant differences in the specific composition of the diets of the five insectivorous flycatchers indicated the differentiation of dietary niches. The study of the gut microbiota and feeding habits of sympatric flycatchers would increase the understanding of the gut microbial diversity of wild birds, and also improve our cognition of the co-evolution and co-adaptation within the host gut microbiota relations.