BACKGROUND: There is a growing interest in the use of hops (Humulus lupulus) as an alternative to antibiotics to manipulate ruminal fermentation. However, the effects of different hop varieties on ruminal fermentation and bacterial populations have not been studied. Here the effects of three hop varieties, Cascade (CAS), Millennium (MIL) and Teamaker (TM), at a level of 800 µg mL−1 inoculum on ruminal fermentation and microbial populations in an artificial rumen system (rusitec) fed a barley silage‐based total mixed ration were investigated. Bacterial populations were assessed using real‐time polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies. RESULTS: All hops reduced (P < 0.001) total gas, methane and the acetate:propionate ratio. Liquid‐associated Fibrobacter succinogenes, Ruminococcus albus and Streptococcus bovis were reduced (P < 0.05) by MIL and TM. Feed particle‐associated S. bovis was reduced (P < 0.01) by MIL and TM, but TM and CAS increased (P < 0.01) Ruminobacter amylophilus and Prevotella bryantii respectively. Methanogens were decreased (P < 0.05) by MIL in both liquid and solid fractions and by CAS in the solid fraction. The total amount of α‐ and β‐acids in hops affected the ruminal fermentation. CONCLUSION: Hop‐induced changes in fermentation and microbial populations may improve energy efficiency use in the rumen. Further research is needed to determine the effects of hops on in vivo ruminal fermentation, microbial populations and animal performance. Copyright © 2012 Society of Chemical Industry