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Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae . As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.