Background: Drought stress is the major environmental stress that affects plant growth and productivity. It triggers in plants a wide range of responses detectable at different scales: molecular, biochemical and physiological levels. At the molecular level the response to drought stress results in the differential expression of several metabolic pathways. For this reason, explore the subtle differences existing in gene expression of drought sensitive and drought tolerant genotypes allows to identify drought-related genes that could be used for selection of drought tolerance traits. Genome-wide RNA-Seq technology was used to compare the drought response of two sorghum genotypes characterized by contrasting water use efficiency. Results: the physiological measurements carried out confirmed the drought sensitivity of IS20351 and the drought tolerance of IS22330 previously studied. The expression of drought-related genes was more abundant in the sensitive genotype IS20351 compared to the tolerant IS22330. The Gene Ontology enrichment highlighted a massive increase in transcript abundance in “response to stress” and “abiotic stimulus”, “oxidation-reduction reaction” in the sensitive genotype IS20351 under drought stress. “Antioxidant” and “secondary metabolism”, “photosynthesis and carbon fixation process”, “lipids” and “carbon metabolism” were the pathways most affected by drought in the sensitive genotype IS20351. The sensitive genotype IS20351 showed under well-watered conditions a lower constitutive expression level of “secondary metabolic process” (GO:0019748) and “glutathione transferase activity” (GO:000004364). Conclusions: RNA-Seq analysis revealed to be a very useful tool to explore differences between sensitive and tolerant sorghum genotypes. The transcriptomic results supported all the physiological measurements and were crucial to clarify the tolerance of the two genotypes studied. The connection between the differential gene expression and the physiological response to drought states unequivocally the drought tolerance of the genotype IS22330 and the strategy adopted to cope with drought stress. Overall design: mRNA profiles of 42-day old leaves of well-watered and drought-stressed Sorghum bicolor plants were generated by deep sequencing, in triplicate, using Illumina GAIIx.