Xylitol is a naturally five -carbon polyol with a high sweetening power. Owing to its physicochemical and technological properties, make it of high value to pharmaceutical, food and chemical industries. The biotechnological method of producing xylitol by microorganisms has been studied as an alternative to the chemical method. This method is of interest because it requires little energy and is very specific. Among the microorganisms, yeasts are considered as the best xylitol producer. In this study xylitol was produced by Rhodotorola mucilaginosa that isolated from leaf of Benjamina. The produced xylitol by R. mucilaginosa was determined and measured by thin layer chromatography, kit and colorimetric methods. This strain produced 6.42 g l-1 xylitol after 48 hours in medium congaing of 40 g l-1 xylose. Consequences of increasing the initial xylose concentration from 60 to 140 g l-1, the final xylitol concentration and yield were also increased. Maximum concentration of produced xylitol by R. mucilaginosa was 49.28 g l-1 (yield of 0.59 g g-1) at 140 g l-1 initial xylose concentration. However further increasing of xylose concentration to 160 g l-1, led to a drastic decrease in xylitol production and yield.