Sunflower stalks can be used for the production of methane, but their recalcitrant structure requires the use of thermo-chemical pretreatments. Two thermal (55 and 170 degrees C) and five thermo-chemical pretreatments (NaOH, H2O2, Ca(OH)(2), HCl and FeCl3) were carried out, followed by anaerobic digestion. The highest methane production (259 +/- 6 mL CH4 g(-1) VS) was achieved after pretreatment at 55 degrees C with 4% NaOH for 24 h. Acidic pretreatments at 170 degrees C removed more than 90% of hemicelluloses and uronic acids whereas alkaline and oxidative pretreatments were more effective in dissolving lignin, However, no pretreatment was effective in reducing the crystallinity of cellulose. Methane production rate was positively correlated with the amount of solubilized matter whereas methane potential was negatively correlated with the amount of lignin. Considering that the major challenge is obtaining increased methane potential, alkaline pretreatments can be recommended in order to optimize the anaerobic digestion of lignocellulosic substrates.