Wheat has remarkable importance among cereals in Egypt. Salt stress affects plant growth, development, and crop productivity. Therefore, salinity tolerance is an essential trait that must be incorporated in crops. This research aimed to investigate molecular and biochemical indicators and defence responses in seedlings of 14 Egyptian wheat genotypes to distinguish the most contrasting salt-responsive genotypes. Analysis of ISSR and SCoT markers revealed high polymorphism and reproducible fingerprinting profiles for evaluating genetic variability within the studied genotypes. The HB-10 and SCoT 1 primers had the highest values for all the studied parameters. All the tested primers generated a set of 66 polymorphic bands among tolerant and sensitive genotypes. The transcript profiles of eight TaWRKY genes showed significant induction under the salinity treatments. Moreover, the expression of TaWRKY6 for genotypes Sids 14 and Sakha 93 sharply increased and recorded the highest expression, while the expression of TaWRKY20 for Misr 1 recorded the lowest expression. Under salt stress, the total sugar, proline, and phenolic contents increased significantly, while the chlorophyll content decreased significantly. Additionally, five peroxidase and polyphenol oxidase isoforms were observed in treated leaves and clustered into five different patterns. Some isoforms increased significantly as salinity levels increased. This increase was clearer in salt-tolerant than in salt-sensitive genotypes. Eighteen protein bands appeared, most of which were not affected by salinity compared with the control, and specific bands were rare. Generally, the Sids 14, Sakha 93, Sohag 4, and Gemmeiza 12 genotypes are considered salt tolerant in comparison to the other genotypes.