Domestication and artificial selection have reduced the level of genetic variation in barley. Inter-specific hybridization is one of the most valuable ways to restore at least part of the lost variation. This study aimed to investigate genetic diversity and screening barley lines which possessed the desired traits, as well as drought tolerance, within two F3 populations derived by crossing a cultivated variety (Reihan 03) with two wild genotypes (HSP21 and HSP45). For this purpose, 162 F3 lines (including 81 lines from each breeding population) were evaluated in three different environments including no stress, moderate drought stress and severe drought stress. The results showed that in both populations, the greatest and smallest variation was obtained for the number of grains per spike and the number of days to physiological maturity, respectively. Mean of the plant height and 100-grain weight in the offspring obtained by crossing with HSP21 were lower than those of the population obtained by HSP45 under the moderate stress environment. Under severe stress environment, the offspring obtained by crossing Reihan 03 with the wild barley genotype HSP45 showed the increase of spike length, number of grains per spike, biological yield and grain yield, and the decrease of the 100-grain weight, as compared to the offspring obtained by crossing Reihan 03 with HSP21. The correlation coefficient analysis for the offspring resulting from crossing Reihan 03 with HSP21 in moderate and severe stress environments showed that grain yield was positively correlated with plant height, biological yield, stress tolerance index (STI) and yield stability index (YSI). The highest grain yield in (HSP21*Reihan 03) derived genotypes, in both control and moderate stress conditions, was revealed by the genotype 584, whereas the highest value in the severe stress condition belonged to the genotype 592. The highest grain yield in (HSP45*Reihan 03) derived genotypes, in the three mentioned environments, was observed for the genotype numbers 689, 671 and 715, respectively. High-yielding lines in both no stress and drought stress conditions were identified by applying multivariate procedures and drought tolerance indices for further studies.