Xylitol is a valuable substance utilized by food and biochemical industries. NAD(P)H-dependent xylose reductase (XR)-encoded by the yeast KmXYL1 gene-is the key enzyme which facilitates reduction of xylose to xylitol. Multi-copy integration of a mutant KmXYL1 (mKmXYL1) gene was carried out using thermotolerant yeast Kluyveromyces marxianus KCTC17555 Delta URA3, in order to enhance xylitol production. After multi-copy integration, the highest xylitol producing strain was isolated and named K. marxianus 17555-JBP2. This strain exhibited 440% higher xylitol production than the parental strain at 30 degrees C. Due to a multi-copy integration of the mKmXYL1 gene, various additional differences between K. marxianus 17555-JBP2 and the parental strain were observed, including a 66% increase in NAD(P)H-dependent XR activity at high temperature (45 degrees C). Quantitative real-time PCR and transcriptome analysis demonstrated that, relative to the parent strain, K. marxianus 17555-JBP2 exhibited two more copies of mKmXY1 genes and a 9.63-fold elevation in transcription of NAD(P)H-dependent XR. After optimization of bioreactor fermentation conditions (agitation speed), high-temperature (40 degrees C) xylitol productivity of K. marxianus 17555-JBP2 exhibited an 81% improvement relative to the parental strain. In this study, we demonstrated that the overexpression of endogenous XR could enhance xylitol productivity at 40 degrees C by thermotolerant K. marxianus.