We describe here a useful metabolic engineering tool to optimize expression levels for multiple genes, Multiple-Gene Promoter Shuffling (MGPS). This method approaches an optimized gene overexpression level by fusing varied strength promoters to genes of interest for a particular pathway. The selection of these promoters is based on the expression levels of the native genes that they control under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation, by shuffling the promoters GND2 and HXK2 for endogenous transaldolase (tal1), transketolase (tkl1) and pyruvate kinase (pyk1) in the Saccharomyces cerevisiae strain FPL-YSX3. This strain has integrated xylose metabolizing genes including xylose reductase, xylitol dehydrogenase and xylulose kinase. The optimal expression levels for these three genes were identified by analysis of the volumetric ethanol production. We found the optimal combination for ethanol production to be GND2-tal1-HXK2-tkl1-HXK2-pyk1. The MGPS method could easily be adapted into other eukaryotic and prokaryotic organisms, to optimize expression of genes for industrial fermentations.