the most important constraints for crop production worldwide. Salinity stress negatively affects the growth and development of wheat leading to reduced grain yield and quality. To address these issues a study was planned to assess the salt tolerance mechanism using physiological (shoot and root length, fresh weight, chlorophyll content) biochemical and molecular-related parameters in four wheat varieties, two tolerant and two sensitive. Wheat seedlings were subjected to three increasing concentrations of salinity stress [(C) 0mM, (T1) 50mM, (T2) 100mM, (T3) 150mM] using NaCl under hydroponic conditions for 21 days. Tolerant wheat genotypes had higher fresh weight and chlorophyll content as compared to sensitive under salt stress. Antioxidant parameters such as hydrogen peroxide (H2O2), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were estimated both in shoot as well as root tissue. Tolerant wheat genotypes induced the antioxidant enzymes CAT and APX more efficiently and hence were more tolerant to the antioxidant stress in the form of H2O2 and MDA. NHK1 and HKT1 genes demonstrated higher expression levels in tolerant varieties. Similarly, proline biosynthesis gene demonstrated enhanced activity in tolerant varieties. The study provides insight into the role of NHX1, HKT1, and P5CS genes in salinity tolerance and their utility as biomarkers for reliable screening for salt tolerance at the seedling stage in wheat genotypes.