Abstract: The gastrointestinal microbiota plays an important role in the growth, nutrition and well being of fishes and it would be necessary to know the microflora structure in the gastrointestinal tract and the influence factors. In this background, we used nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis to investigate microbiota in the intestine of bighead carp and paddlefish. In this research, bighead carp was fed with live feed (BI-L) and compound feed (BI-C), and paddlefish was fed with live feed (PI-L, PS-L) and compound feed (PI-C, PS-C). A total of 16 bacterial species were identified by phylogenetic analysis. The Firmicutes group (31.25%, 5 OTU) totally dominated the bacterial communities in bighead carp and paddlefish. The other identified bacterial species included -Proteobacteria (25%, 4 OTU), Bacteroidetes (12.5%, 2 OTU), Fusobacteria (12.5%, 2 OTU), Cyanobacteria (6.25%, one OTU), Actinobacteria (6.25%, one OTU) and -Proteobacteria (6.25%, one OTU). The higher similarities (above 49% in paddlefish and 59% in bighead carp) of the DGGE band patterns in intestinal bacteria between two groups feeding with different feed indicated that there are more stable microbial communities existed in these specifically ecological niches in both bighead carp and paddlefish. However, the microflora structure in the stomach has considerable difference in the two groups of paddlefish feeding with different feed, probably indicating that the community structures in stomach of paddlefish were affected by diet easily and significantly.