Salinity is a widespread abiotic stress that devastatingly impacts wheat growth and restricts its productivity worldwide. The present study is aimed at elucidating biochemical, physiological, anatomical, gene expression analysis, and agronomic responses of three diverse wheat genotypes to different salinity levels. A salinity treatment of 5000 and 7000 ppm gradually reduced photosynthetic pigments, anatomical root and leaf measurements and agronomic traits of all evaluated wheat genotypes (Ismailia line, Misr 1, and Misr 3). In addition, increasing salinity levels substantially decreased all anatomical root and leaf measurements except sclerenchyma tissue upper and lower vascular bundle thickness compared with unstressed plants. However, proline content in stressed plants was stimulated by increasing salinity levels in all evaluated wheat genotypes. Moreover, Na+ ions content and antioxidant enzyme activities in stressed leaves increased the high level of salinity in all genotypes. The evaluated wheat genotypes demonstrated substantial variations in all studied characters. The Ismailia line exhibited the uppermost performance in photosynthetic pigments under both salinity levels. Additionally, the Ismailia line was superior in the activity of superoxide dismutase (SOD), catalase activity (CAT), peroxidase (POX), and polyphenol oxidase (PPO) enzymes followed by Misr 1. Moreover, the Ismailia line recorded the maximum anatomical root and leaf measurements under salinity stress, which enhanced its tolerance to salinity stress. The Ismailia line and Misr 3 presented high up-regulation of H+ATPase, NHX2 HAK, and HKT genes in the root and leaf under both salinity levels. The positive physiological, anatomical, and molecular responses of the Ismailia line under salinity stress were reflected on agronomic performance and exhibited superior values of all evaluated agronomic traits.