The potential of wheat straw hydrolysate for biogas production was investigated in continuous stirred tank reactor (CSTR) and up-flow anaerobic sludge bed (UASB) reactors. The hydrolysate originated as a side stream from a pilot plant pretreating wheat straw hydrothermally (195 °C for 10–12 min) for producing 2nd generation bioethanol [Kaparaju, P., Serrano, M., Thomsen, A.B., Kongjan, P., Angelidaki, I., 2009. Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept. Bioresource Technology 100 (9), 2562–2568]. Results from batch assays showed that hydrolysate had a methane potential of 384 ml/g-volatile solids (VS)added. Process performance in CTSR and UASB reactors was investigated by varying hydrolysate concentration and/or organic loading rate (OLR). In CSTR, methane yields increased with increase in hydrolysate concentration and maximum yield of 297 ml/g-COD was obtained at an OLR of 1.9 g-COD/l d and 100% (v/v) hydrolysate. On the other hand, process performance and methane yields in UASB were affected by OLR and/or substrate concentration. Maximum methane yields of 267 ml/g-COD (COD removal of 72%) was obtained in UASB reactor when operated at an OLR of 2.8 g-COD/l d but with only 10% (v/v) hydrolysate. However, co-digestion of hydrolysate with pig manure (1:3 v/v ratio) improved the process performance and resulted in methane yield of 219 ml/g-COD (COD removal of 72%). Thus, anaerobic digestion of hydrolysate for biogas production was feasible in both CSTR and UASB reactor types. However, biogas process was affected by the reactor type and operating conditions.