Lowland and upland rice, as for two ecotypes, that have been exhibited different tolerance levels under hypoxia conditions. However, the molecular mechanisms underlying rice root hypoxia tolerance between them are not fully understood. This study was performed to assess the morphological, physiological and transcriptional changes of roots in one lowland rice YueFu (YF) and one upland rice IRAT109 (IR) genotype. A morpho-physiological analysis revealed that compared to IR, YF showed less reduction of root length, root and shoot biomass, formed more aerenchyma in the root, and kept more oxygen influxes in root under hypoxia conditions. Indole-3-acetic acid (IAA) fluxes patterns exhibited a different response to hypoxia in YF and IR. The contents of IAA, ethylene and hydrogen peroxide were significantly increased in YF and IR, but nitric oxide significantly increased only in YF under hypoxic conditions. Subsequently, transcriptome analysis revealed that more differentially expressed genes (DEGs) were identified to respond to hypoxia in YF than IR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that DEGs in both genotypes enriched in energy metabolism, aerenchyma formation, reactive oxygen species, and cell wall modification, whereas more related DEGs in YF significantly enriched in these pathways than IR. The specific DEGs in YF especially enriched in phytohormone metabolism and signaling, such as auxin, jasmonic acid, and ethylene, but the specific DEGs in IR especially enriched in photosynthesis. All these results demonstrate that YF is more tolerant to hypoxia than IR, and elucidate some specific mechanisms underlying the differential hypoxia tolerance in lowland and upland rice. This study has provided valuable candidate genes for genetic improvement of rice in adapting to hypoxia stress.