SALINITY stress significantly impacts the growth, development and yield of sorghum. Knowledge about the productivity of sorghum under salinity stress condition is lagging, due to the marginally limited information about the physio-biochemical and molecular mechanisms underpinning salinity stress tolerance. The study aimed to investigate the physio-biochemical and molecular mechanisms associated with the differential responses under salinity stress condition in sorghum. To achieve our objectives, the physiological and biochemical salinity stress parameters, including membrane stability index, enzyme antioxidant activities, proline and chlorophyll contents, measured at the seedling stage, were elucidated to identify the salinity stress response status of the genotypes. The quantitative real-time polymerase chain reaction (qPCR) was perform to elucidate the expression pattern of different categories of genes under salinity stress in sorghum genotypes. From our results, enhanced enzyme antioxidant activities, membrane status, increased proline content and lower K+/Na+ concentration under 300mM NaCl stress identified PI 585451 to be the most salinity stress-tolerant genotype. Nevertheless, increased MDA and Na+/K+ level, lower proline, chlorophyll content and antioxidant enzyme activities in PI 585454 marked it to be sensitive to salinity stress. Besides, the transcript expression analysis of different genes, showed an upregulation in PI 585451 than in PI 585454. The results highlighted the differences in metabolic response to salinity stress that may play an important role in the provision of information, required for breeding and development of sorghum genotypes that are tolerant to salinity stress.