This study evaluated the biotechnological production of xylitol from sorghum forage biomass. The yeast Candida guilliermondii was cultivated in hemicellulosic hydrolysates obtained from biomass of three sorghum varieties (A, B, and C). First, the biomass was chemically characterized and subjected to dilute acid hydrolysis to obtain the hemicellulosic hydrolysates which were vacuum-concentrated and detoxified with activated charcoal. The hemicellulosic hydrolysates (initial pH 5.5) were supplemented with nutrients, and fermentations were conducted in 125-mL Erlenmeyer flasks containing 50 mL medium, under 200 rpm, at 30 °C for 96 h. Fermentations were evaluated by determining the parameters xylitol yield (Y P/S ) and productivity (QP), as well as the activities of the enzymes xylose reductase (XR) and xylitol dehydrogenase (XDH). There was no significant difference among the three varieties with respect to the contents of cellulose, hemicellulose, and lignin, although differences were found in the hydrolysate fermentability. Maximum xylitol yield and productivity values for variety A were 0.35 g/g and 0.16 g/L.h(-1), respectively. It was coincident with XR (0.25 U/mg prot) and XDH (0.17 U/mg prot) maximum activities. Lower values were obtained for varieties B and C, which were 0.25 and 0.17 g/g for yield and 0.12 and 0.063 g/L.h(-1) for productivity.