In order to investigate the differences of intestinal microflora of freshwater fishes in two regions of China, we chose grass crap（Ctenopharyngodon idella）, crucian crap（Carassius auratus) and crap（Cyprinus carpio）from Yucheng city (in Shandong Province belonging to northern China) and Wangcheng District (in Hunan Province belonging to southern China) as the experimental objects. The intestinal contents of fishes were sequenced by 16S rRNA gene V4 hypervariable regions via S5TM XL platform and then the sequencing data were analyzed. The results revealed that the dominant intestinal bacteria of grass crap, crucian crap and crap were Proteobacteria, Fusobacteria, Firmicutes and Bacteroidetes at phylum category. The sum of the four kinds of bacteria in each group was more than 80%. Based on the rank of BrayCurtis distance, the differences between groups were significant. Compared with crap group, the differences among groups were significantly greater than that within groups (R=0.307, P<0.05), speculating that the difference of characteristic flora is related to the influence of environment. LDA （linear discriminant analysis）analysis showed that the biomarker of Hunan crap group was Vibrio. Meanwhile, it was Clostridium in Shandong crap. The statistical analysis of harmful strains showed that the relative abundance of Aeromonas hydrophila in Hunan grass crap group and Hunan crucian crap group was significantly higher than that in Shandong groups, and the relative abundance of Vibrio in Shandong crap group and Shandong crucian crap group was significantly higher than that in Hunan groups. The ability of fishes in different areas to resist the invasion of foreign harmful bacteria will change with the environment. The comparison and analysis of the intestinal microbial community structure of freshwater fishes in the two areas are established for the further research of freshwater fish growth.