BACKGROUND Reportedly, various mixes of seeds were quasi-randomly selected to startup anaerobic digesters. In contrast, this study examines the impact of inoculating thermophilic anaerobic digesters with a designed mix of non-acclimated seeds based on their methanogen composition, using Quantitative Polymerase Chain Reaction (QPCR) of 16S rRNA gene, to achieve high abundance and diversity of methanogens. RESULTS Based on QPCR results, two seed mixes were selected to inoculate two anaerobic digesters: digester (A) was inoculated with a control seed consisting of digestate, manure, and activated sludge; and digester (B) was inoculated with a further methanogen-enriched seed consisting of the control seed with added compost and leachate. Both seed combinations yielded a balanced microflora that is able to achieve a successful startup. However, upon reaching steady state, digester B exhibited lower propionate levels, resulting in lower VFA concentration and increased buffering capacity, indicating greater stability. Acetotrophs and hydrogenotrophs were dominated by Methanosarcinaceae and Methanobacteriales, respectively, in both digesters, exhibiting an average ratio of 66-to-34% in A and 76-to-24% in B during steady state. CONCLUSION The inoculation strategy in digester B resulted in improved stability, lower propionate concentration and 10% higher relative abundance of acetotrophs.