Dietary changes can alter the rumen environment and provoke shifts in microbial communities leading to incomplete biohydrogenation (BH). The objective of this study was to compare bacterial diversity in diets previously shown to cause shifts in BH intermediates. Diets containing low (LF) or high (HF) concentrations of unsaturated fatty acids (0 or 3.3% soybean oil added) were modified using corn sources with low (L), medium (M) or high (H) starch degradability (SKd; 48.4 L, 66.2 M, or 84.0% h⁻¹ in 7 h in vitro test) and arranged in a 2 × 3 factorial design. Diets were fed for 4 10 d periods.Bacterial community composition from overflow samples was determined using Illumina MiSeq16S rRNA gene V4 variable region amplicon sequencing. Significant differences in β diversity among sample groupings were determined using a Python script within QIIME to perform a PERMANOVA. For individual relative abundance (rA) of interest, the MIXED procedure of SAS was used. Significance for main effect of fat and linear and quadratic contrasts for SKd were set at P ≤ 0·10. Results showed 519 species belonging to 248 genera across treatments. Beta diversity was different between LF and HF (P = 0.10); and significantly increased with SKd (P = 0.02) with a pronounced separation between L and H SKd (P < 0.01). Among the 60 species of most frequently detected taxa, 29 showed a progressive decrease (n = 20) or increase (n = 9) in frequency moving from L to H SKd respectively. The rA of fibrolytic bacteria Prevotella ruminicola was increased while Ruminococcus flavefaciens was reduced (P ≤ 0.01) as fat was added. There was a linear decrease in rA of Butyvibrio fibrisolvens and Butyvibrio hungatei (BH enabler; P ≤ 0.10) as SKd increased. Whereas Streptococcus bovis and Prevotella bryantii (amylolytic) showed a linear increase as Skd increased (P ≤ 0.05). Taxa responsible for lipolysis (Anaerovibrio lipolyticus) or utilizing lactic acid (Megasphaera elsdenii) were not different. These results suggest that fat level affect bacteria diversity and that increasing the SKd with constant starch level causes significant changes in microbial communities.