C-13 metabolic flux analysis ((13)CMFA) has been extensively applied in studying the glucose metabolism of yeast strains such as Saccharomyces cerevisiae and Scheffersomyces stipitis. Here, we tried to augment the previous fluxomic studies by applying (13)CMFA to rigorously investigate the metabolic flux distributions in S. stipitis and the recombinant S. cerevisiae strains when xylose was utilized as the sole carbon substrate. It was found that less carbon fluxes were diverted into the TCA cycle in S. stipitis than the recombinant S. cerevisiae strains. Compared to single sugar utilization, the co-utilization of glucose and xylose by S. stipitis led to increased metabolic fluxes into the futile pathway and the TCA cycle, but did not improve sugar-based ethanol yield. In addition, heterologous expression of xylose pathway in engineered S. cerevisiae strains may affect the glucose utilization. (c) 2013 American Institute of Chemical Engineers AIChE J, 59: 3195-3202, 2013