ABSTRACT Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering ( P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing ( P ≤ 0.05) among diets. During acidosis, levels of Atopobium , Desulfocurvus , Fervidicola , Lactobacillus , and Olsenella increased, while during the recovery, Desulfocurvus , Lactobacillus , and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed ( P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium , cc142, Lactobacillus , Olsenella , RC39, Sharpea , Solobacterium , Succiniclasticum , and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.