Soil salinity is increasing due to several factors such as climate change and areas with uneven rainfall. This increase in level of salinity compelled the cotton breeders to develop a new germplasm that exhibit the suitable for salty soil. This study aimed to determine the salt tolerance of 50 accessions of Gossypium hirsutum in hydroponic conditions having three levels of NaCl, i.e., 0 mM, 150 mM, and 200 mM. The experiment was carried out in a completely randomized design with a factorial arrangement. Morphological, physiological, and biochemical attributes were estimated in these genotypes. The Na+/K+ ratio was determined by dry digestion method. Salt-susceptible and -tolerant genotypes were identified by biplot and cluster analysis. The genotypes showed significant differences for morphophysiological and biochemical parameters. In control, Cyto-515 showed enhanced growth with shoot length (30.20 cm), root length (20.63 cm), fresh shoot weight (2.34 g), and fresh root weight (0.93 g), while under 150 mM and 200 mM salinity levels, MNH-992 had the maximum root length (15.67 cm) and shoot length (24.67 cm). At a 150 mM salinity level, maximum levels of antioxidants were found in Kehkshan and CIM-595, while at a 200 mM salinity level, AA-703, CIM-595, and Kehkshan showed maximum values of antioxidants. The highest Na+/K+ ratio was observed in VH-363 and FH-114, while Kehkshan had lowest Na+/K+ ratio. The biplot analysis revealed that Kehkshan, CIM-595, VH-330, Cyto-178, MNH-992, and Cyto-515 were widely dispersed and distant from the origin, and exhibiting variability for morphophysiological and biochemical traits under the salt stress. In terms of performance across the treatments, accessions MNH-992, Kehkshan, Cyto-515, and CIM-595 performed significantly better. Peroxidase activity, proline contents, H2O2 determination, and Na+/K+ ratio were shown to be useful for the salt tolerance selection criteria. The potential of such salt tolerant accessions (MNH-992, Kehkshan, Cyto-515, and CIM-595) could be assessed after planting in salt affected areas and could be used in breeding programs for the development of diverse salt tolerant new genotypes of upland cotton.