Xylose derived from lignocellulose can be utilized to produce ethanol and other high-value chemicals, such as xylitol. The xylitol production through fermentation of lignocellulosic hydrolysate by microorganisms offers advantages of high product yield, high selectivity, and efficacy in mild conditions. In this study, non-detoxified hemicellulose hydrolysate from napiergrass was used for xylitol production by a recombinant flocculating strain of Saccharomyces cerevisiae. An optimization study was conducted with the strain at 35 °C. A promising xylitol yield of 0.96 g/g xylose with no addition of glucose required during the fermentation process, which suggests an extensive potential improvement for the economics of lignocellulosic xylitol production.