Background Brazil is the biggest ethanol producer in the world using sugarcane as substrate. From this process, bagasse is the main resulting by-product which can be used to produce second generation ethanol. Although Saccharomyces cerevisiae is the ideal yeast for the fermentative process, it cannot ferment xylose, one of the most abundant sugars in lignocellulosic biomass. Different yeast can ferment xylose, including S. stipitis [1], but not with the same efficiently rate as S. cerevisiae. Genes from xylose-fermenting yeasts can be used to genetically engineer S. cerevisiae to improve bioethanol production. Since it is widely known that the transport of sugars inside the cells is a limiting factor for the fermentation of sugars, and several reports have demonstrate that an increase in transport activity also increases the fermentative capacity of yeast cells [2], in the present work we have used a S. cerevisiae strain lacking hexose transporters (hxt-null) to screen a S. stipitis genomic library to identify putative xylose transporter genes.