Coniochaeta ligniaria C8100 is a xylose non-utilizing mutant derived from wild-type (WT) C. ligniaria NRRL30616. While NRRL30616 metabolizes xylose as a source of carbon and energy, C8100 does not grow on xylose and has reduced xylose reductase (XR) and xylitol dehydrogenase activities compared to WT. Instead, the mutant converts a portion of xylose to xylitol. The use of this mutant strain was investigated to identify culture conditions that affect xylitol production. Optimal production of xylitol by C. ligniaria occurred at 30-32 °C and pH 5-8 in yeast-peptone medium. C8100 produced up to 0.64 g xylitol/g xylose in culture medium and 0.34 g xylitol/g xylose in corn stover dilute acid hydrolysate. As observed in other fungi, productivity was much lower than values reported for yeast. Addition of benzoate, a chaotropic stressor thought to induce production of polyols as a stress response, increased xylitol production. Expression of the Scheffersomyces stipitis XYL1 gene in C. ligniaria C8100 restored XR activity in the mutant to near-WT levels and increased xylitol yield by 20% in rich medium and 11% in corn stover dilute acid hydrolysate. Like the WT strain, C. ligniaria C8100 metabolizes inhibitors in biomass hydrolysates and this inhibitor-tolerant strain may have useful properties for production of xylitol from lignocellulosic biomass.