Abstract In the present study, thermophilic anaerobic digestion of wheat straw stillage was investigated. Methane potential of stillage was determined in batch experiments at two different substrate concentrations. Results showed that higher methane yields of 324 ml/g-(volatile solids) VS added were obtained at stillage concentrations of 12.8 g-VS/L than at 25.6 g-VS/l. Continuous anaerobic digestion of stillage was performed in an up-flow anaerobic sludge blanket (UASB) reactor at 55 °C with 2 days hydraulic retention time. Results showed that both substrate concentration and organic loading rate (OLR) influenced process performance and methane yields. Maximum methane yield of 155 ml CH 4 /g-COD was obtained at stillage mixtures with water of 25% (v/v) in the feed and at an OLR of 17.1 g-COD/(l.d). Soluble chemical oxygen demand (SCOD) removal at this OLR was 76% (w/w). Increase in OLR to 41.2 g-COD/(l.d) and/or stillage concentration in the feed to 33–50% (v/v) resulted in low methane yields or complete process failure. The results showed that thermophilic anaerobic digestion of wheat straw stillage alone for methane production is feasible in UASB reactor at an OLR of 17.1 g-COD/(l.d) and at substrate concentration of 25% in the feed. The produced methane could improve the process energy and economics of a bioethanol plant and also enable to utilize the stillage in a sustainable manner.