Aims: To reveal the effects of fish genotype, feeding habits and serum physiological index on the composition of gastrointestinal microbiota, eight fish species with four different feeding habits were investigated. Methods and Results: The V1 to V3 regions of 16S rRNA gene were analysed by high-throughput sequencing (454 platform) to compare the gut microbiota of different fish species. A total of 551 995 high-quality sequences with an average length of 463 bp were obtained from the 48 samples. No significant difference was observed among the detected sequences obtained from fishes with different feeding habits (One-way ANOVA, F = 1Á003, P = 0Á400), but the number of OTUs among different feeding habits was significantly different (One-way ANOVA, F = 7Á564, P < 0Á001). Additionally, significant correlations were detected between the fish genotype and microbial composition (partial Mantel test, all P values = 0Á001) in the stomach, foregut and hindgut. Moreover, different core intestinal microbiota was also noticed in the eight fish species with different feeding habits. Conclusions: Feeding habits and genotype clearly affected the gastrointestinal microbiota of fish. Moreover, the evolutionary process shaped the serum physiological indexes of fish. Significance and Impact of the Study: This study provided much important information for developing commercial fish feeds.