Xylitol, a naturally occurring five-carbon sugar alcohol derived from D-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use D-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the D-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (∆xdh1) and a double mutant where additionally L-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (∆lad1∆xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % D-xylose by the ∆xdh1 and ∆lad1∆xdh1 strains, respectively.