Nowadays, water scarcity is a great danger to agriculture development, in semiarid and arid regions beside climate change risks. Egypt is suffering from scarce water resources for agriculture; it is of high priority to rationalize irrigation water use. During two seasons, 2019/2020 and 2020/2021 the experimental were carried out to investigate the effect of three different irrigation water treatments (I1 at 45, I2 at 60 and I3 at 75% depletion of available soil moisture) on some photosynthetic, agronomical, grain quality parameters, water productivity for eight barley genotypes. Also, classify them on the SSR molecular level. The results showed that there were high genetic variations found among the eight genotypes with significant responses to irrigation water treatments. Rationalize irrigation water from I1 to I3 had a negative effect on all studied phenotypic traits, whereas had an appositive effect on leaf diffusive resistance and cured protein content and inducement all genotypes to flower early by average increasing (27.7, 12.7 and 7.08%) respectively. Giza 138, Giza 131, and Line 4 gave high mean performance values of the measured characters besides, attaining high WP values were 1.14, 1.08, and 0.89 kg grain/m3 applied water I1, I2, and I3 irrigation treatments, respectively. While, WUE values increased with increasing water availability. Water use efficiency values were 1.92, 1.71, and 1.34 kg grain/m3 consumed water for I1, I2, and I3 irrigation treatments, respectively. Twenty-six alleles were generated using ten SSR primers with a mean value of 2.6 alleles per locus. The Polymorphism Information Content (PIC) value of each SSRs marker ranged from 0.33 (Bmag 0387) to 0.47 (Bmac 0167) with an average value of 0.34. Cluster analysis clustered the eight barley genotypes into two major clusters divided according to their response to water stress tolerance. The genetic information about eight barley genotypes for water stress tolerance was established, for use them in breeding programs in Egypt.