The macroarray methodology was used in this work aiming the identification of gene expression under two water stress conditions in sugarcane plants. We evaluated the genes expressed in two sugarcane cultivars considered as tolerant to stress (SP83-5073 and SP83-2847) through the 3575 ESTs of leaves selected in SUCEST database. The studied genes were analyzed by the R statistical program based in the Limma functions. Genes were classified by its statistical significance level and for both cultivars a hundred genes were selected. The SP83-2847 cultivar showed differentially expressed genes just after 24 hours under water stress conditions (mild), while for the SP83-5073 cultivar the expression level for the analyzed genes was modified preferentially at severe stress conditions, what may represent a different level of water stress tolerance between cultivars. Fifteen induced genes were common for two cultivars, such as kinase proteins and the proteins involved in the poliamine metabolism. In these selected genes four presented no similarity with described proteins in the searched databases. Previously described genes, important for water stress response, as membrane integral proteins (MIP), ABC transporters, heat shock proteins and proteins involved to antioxidant defense were also observed. The macroarray results have been confirmed with RT-PCR. Genes with no similarity with known proteins are being analyzed for the presence of motives and domains that can help its characterization. These genes can be very useful therefore they may represent the discovery of new information. The characterization of genes that answer to water stress in different tolerant genotypes can supply new information on the mechanism of defense of these plants.