Real time PCR was used in this study to determine the effect of triticale dried distillers grains with solubles (TDDGS) as a replacement for grain or barley silage in finishing diets on the presence of six classical ruminal bacterial species (Succinivibrio dextrinosolvens, Selenomonas ruminantium, Streptococcus bovis, Megasphaera elsdenii, Prevotella ruminicola and Fibrobacter succinogenes) within the rumen contents of feedlot cattle. This study was divided into a step-wise adaptation experiment (112 days) that examined the effects of adaptation to diets containing increasing levels of TDDGS up to 30% (n = 4), a short-term experiment comparing animals (n = 16) fed control, 20%, 25% or 30% TDDGS diets over 28 days, and a rapid transition experiment (56 days) where animals (n = 4) were rapidly switched from a diet containing 30% TDDGS to a barley-based diet with no TDDGS. It was found that feeding TDDGS as replacement for barley grain (control vs. 20% TDDGS) decreased 16S rRNA copy numbers of starch-fermenting S. ruminantium and S. bovis (p<0.001 and p = 0.04, respectively), but did not alter 16S rRNA copy numbers of the other rumen bacteria. Furthermore, feeding TDDGS as a replacement barley silage (20% vs. 25% and 30% TDDGS) increased 16S rRNA copy numbers of S. ruminantium, M. elsdenii and F. succinogenes (p<0.001; p = 0.03 and p<0.001, respectively), but decreased (p<0.001) the 16S rRNA copy number of P. ruminicola. Upon removal of 30% TDDGS and return to the control diet, 16S rRNA copy numbers of S. ruminantium, M. elsdenii and F. succinogenes decreased (p = 0.01; p = 0.03 and p = 0.01, respectively), but S. dextrinosolvens and S. bovis increased (p = 0.04 and p = 0.009, respectively). The results suggest that replacement of TDDGS for grain reduces 16S rRNA copy numbers of starch-fermenting bacteria, whereas substitution for barley silage increases 16S rRNA copy numbers of bacteria involved in fibre digestion and the metabolism of lactic acid. This outcome supports the contention that the fibre in TDDGS is highly fermentable.