Xylose from hemicellulose hydrolyzates (HH) offers good prospects for valorization since it can be converted into a variety of high value-added products if it is separated and purified from the side streams of pulping. The influence of hydrothermal pretreatment of organosolv HH from beechwood pulping on the separation of xylose by four nanofiltration membranes was investigated. At first, a hydrothermal process was developed using response surface methodology to study appropriate process parameters (temperature and residence time) for maximum conversion of hemicellulose to xylose. Then, the nanofiltration process using a model solution, HH, and hydrothermally treated HH was assessed by an extensive performance and fouling analysis. Atomic force microscopy and physisorption analysis were applied to characterize the membranes NF (Alfa Laval), DK (GE Osmonics), TS40 (TriSep), and NF90 (DOW). Hydrothermal conversion at 180 degrees C and 3.1 min led to a conversion of almost 100% of hemicellulose to xylose without further degradation to chemical successors. The hydrothermal pretreatment of HH had positive effects on membrane performance and reduces fouling. Membrane characteristics such as lower surface roughness, a larger pore width and specific surface area, and smaller contact angles favored a higher flux and the ability to separate inhibitors as well as reduced fouling. Alkaline cleaning with P3-Ultrasil-53 was sufficient to remove the foulants and maintain a stable pure water flux.