Sorghum [Sorghum bicolor (L.) Moench] is considered as an important staple crop in the tropical regions. However, the productivity of this useful crop is hindered by drought which contributes to significant yield reduction. The present study aimed to decipher the effects of drought stress on physiological, biochemical and gene expression changes in sorghum genotypes and to ascertain the differences in their response to drought stress. To achieve these objectives, six sorghum genotypes were grown in pot culture in a greenhouse, in a randomized complete block design and exposed to water stress treatment for 10 days. From the study, drought stress caused a significant (P < .05) reduction in plant height, leaf water and chlorophyll contents while the proline, malondialdehyde (MDA), soluble sugar, electrolyte leakage (EL), hydrogen peroxide (H2O2) and antioxidant enzymes activity increased significantly (P < .05) and differentially in all sorghum genotypes. Among the genotypes investigated, PI 585456 showed enhanced performance and was considered as the most tolerant to drought in relation to plant growth and water relation, membrane status, photosynthetic activity, ROS and osmolytes accumulation and antioxidant enzymes activity. Furthermore, the transcript expression analyses of different categories of drought-responsive genes, viz; antioxidant-related, osmolytes biosynthesis-related, dehydrin-related, photosystem-related and transcription-related were differentially upregulated in sorghum genotypes investigated. The results revealed the differences in metabolic response to drought among the genotypes, which accentuated the physiological, biochemical and molecular mechanism related to a specific response that may play a vital role in drought tolerance in sorghum.