Analysis of the transcriptomic changes produced in response to hypoxia in root tissues from two rootstock Prunus genotypes differing in their sensitivity to waterlogging: resistant Myrobalan 'P.2175' (P. cerasifera Erhr.), and sensitive 'Felinem' hybrid [P. amygdalus Batsch × P. persica (L.) Batsch] revealed alterations in both metabolism and regulatory processes. Early hypoxia response in both genotypes is characterized by a molecular program aimed to adapt the cell metabolism to the new conditions. Upon hypoxia conditions, tolerant Myrobalan represses first secondary metabolism gene expression as a strategy to prevent the waste of resources/energy, and by the up-regulation of protein degradation genes probably leading to structural adaptations to long-term response to hypoxia. In response to the same conditions, sensitive 'Felinem' up-regulates a core of signal transduction and transcription factor genes. A combination of PLS-DA and qRT-PCR approaches revealed a set of transcription factors and signalling molecules as differentially regulated in the sensitive and tolerant genotypes including the peach orthologs for oxygen sensors. Apart from providing insights into the molecular processes underlying the differential response to waterlogging of two Prunus rootstocks, our approach reveals a set of candidate genes to be used expression biomarkers for biotech or breeding approaches to waterlogging tolerance.