The physiological and molecular responses to water stress are mediated by a range of mechanisms, many of which involve abscisic acid (ABA)-dependent signaling pathways. In addition, plants contain drought response genes that can be induced by ABA-independent routes, mediated by secondary messengers such as Ca 2+ , or regulated by epigenetic modifications. The complex processes involved in the response to water stress can be investigated using molecular techniques to evaluate the expression patterns of genes of interest and to infer the behavior of different genotypes and species. In the present study, we first analyzed the stability of a set of reference genes for normalization of the gene expression with real-time quantitative polymerase chain reaction (RT-qPCR), since there were no results related to the genotype used in this study. We verified that although there were some variations between algorithms used, the three most stable reference genes were SAND, PP2A-3 and EF-1α. The expressions of genes encoding for proteins associated with drought-tolerance responses, namely 9- cis -epoxycarotenoid dioxygenase 3 (EgrNCED3), pyrabactin resistance 1 (EgrPYR1), dehydration-responsive element-binding 2.5 (EgrDREB2.5) transcription factors, calcium-dependent protein kinase 26 (EgrCDPK26), methyl transferase 1 (EgrMET1) and deficient in DNA methylation 1 (EgrDDM1) protein, were determined by RT-qPCR in leaf samples from drought sensitive (VM05) and drought tolerant (VM01) clones of the hybrid Eucalyptus camaldulensis x Eucalyptus urophylla grown under water stress and irrigation conditions. When the two clones were maintained under conditions of water deficiency, VM01 exhibited higher expression levels of EgrNCED3 and EgrPYR1 genes than VM05 at all sampling times, implying that ABA biosynthesis and subsequent induction of the ABA-dependent cascade mediated by the PYR1-ABA receptor complex were enhanced in the tolerant clone. Under water-stress conditions, this clone also presented increased expression of the EgrDREB2.5 gene, representative of an ABA-independent cascade, and of the EgrCPK26 gene, related to stomatal opening and closure. On the other hand, the expression levels of EgrMET1 and EgrDDM1 genes in the sensitive clone were higher than in the tolerant clone under all conditions, showing a putative impact of epigenetic modifications on tolerance to water deficiency. The results obtained indicate that the superior ability of the VM01-tolerant clone to perceive water deficiency and activate drought-resistance genes is associated with the high expression levels of EgrNCED3, EgrPYR1 and EgrDREB2.5 under water-stress conditions. These findings will facilitate future research on the functional characterization of stress-related response genes, the identification of molecular markers, the evaluation of drought tolerance and genetic transformation in tree species. [ABSTRACT FROM AUTHOR]