Aims: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture- dependent conventional and independent molecular techniques. Methods and Results: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. Conclusions: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. Significance and Impact of the Study: The fish gut harbours a larger bacterial diversity,than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.