16S rRNA gene-based analysis of rumen Prevotella was carried out to estimate the diversity and diet specificity of bacteria belonging to this genus. Total DNA was extracted from the rumen digesta of three sheep fed two diets with different hay-to-concentrate ratios (10 : 1 and 1 : 2). Real-time PCR quantification of Prevotella revealed that the relative abundance of this genus in the total rumen bacteria was up to 19.7%, while the representative species Prevotella bryantii and Prevotella ruminicola accounted for only 0.6% and 3.8%, respectively. Denaturing gradient gel electrophoresis analysis for Prevotella revealed shifts in the community composition with the diet. Analysis of 16S rRNA gene clone libraries showed significant differences (P=0.001) between clones detected from the sheep on the diets with different hay-to-concentrate ratios. The majority (87.8%) of Prevotella clones had <97% sequence similarity with known rumen Prevotella. These data suggest that uncultured Prevotella is more abundant than known Prevotella and that members of this genus appear to have specific metabolic niches.