Root hypoxia in fruit trees affects growth, vegetative development, and reproductive development, which is reflected in low productivity, poor fruit quality, and premature decay of trees. Using Illumina Hiseq2000, we performed transcriptome analysis of roots from two different rootstocks, ‘Mariana 2624’ and ‘Mazzard F12/1,’ which are tolerant and sensitive to hypoxia, respectively. Transcriptomes from control and hypoxia-stressed plants (6, 24, and 72 h) were compared, using Prunus persica (L.) as reference genome. Hypoxic conditions altered the transcription in both genotypes. There were a high number of common differentially expressed genes (DEG) between the two genotypes for each sampling time, but also exclusive DEG for each genotype, with a few DEG that presented opposite modes of regulations during the hypoxia treatment. An important group of DEGs exclusively upregulated in the tolerant genotype are associated to enzymes of posttranslational protein modifications, such as leucine-rich repeat (LRR), kinases and ubiquitin-protein ligases, regulation of transcription, and process of oxide reduction. Singular enrichment analysis of gene ontology (GO), detected at least 115 GOs involved in the response to root hypoxia in the sensitive and/or tolerant genotypes. At least 25 GOs were identified as part of the baseline differences between the genotypes, most GO were disturbed in the sensitive genotype. The contribution from the baseline gene expression to the differential response between the Prunus genotypes is evidence that the resistant genotype is already “prepared” for a hypoxia event. An example are GO BP:0042221 of response to chemical stimulus; BP:0006979 of response to oxidative stress; MF:0016209 of antioxidant activity; MF:0016684 of oxidoreductase activity, acting on peroxide as acceptor; and MF:0004601 of peroxidase activity, which were disturbed only in the sensitive genotype, but not in the tolerant.