This paper presents the results of an experimental methane fermentation study on untreated, NaOH and hydrothermal pretreated substrates of wheat straw. Experiments were conducted at 37 °C (mesophilic temperature). Substrates concentration were maintained at 4.45% VS (44.5 g VS/L). Untreated wheat straw substrate had resulted into specific methane and biogas production yields of 78.4 L/kg VSa and 188.4 L/kg VSa, respectively. The specific methane and biogas production yield of NaOH pretreated wheat straw substrate had resulted into 165.9 L/kg VSa and 353.2 L/kg VSa, respectively. Hydrothermal pretreated wheat straw substrate had resulted to yield specific methane and biogas production of 94.1 L/kg VSa and 205.7 L/kg VSa, respectively. NaOH pretreated substrate produced 87.5% higher biogas production and 111.6% higher methane production compared to the untreated wheat straw substrate. Hydrothermal pretreated substrate had resulted into an increase of 9.2% in biogas production and 20.0% in methane production compared to that of untreated wheat straw substrate. ► NaOH, hydrothermal pretreated substrates of wheat straw were studied for Methane fermentation. ► Minimum 5% NaOH addition is required for hydrothermal substrate to maintain a suitable pH. ► Biogas and methane production were increased by 87.5% and 111.6% for 4% NaOH pretreated substrate. ► Wheat straw NaOH pretreatment found a potential method to enhance biogas and methane production.