There are contrasting reports on the relationship between yield and drought tolerance of crops with root characteristics. This research aimed to study grain yield and root-related traits (at two depths) under optimal and drought stress conditions and assess the effect of root-related traits on grain yield and drought tolerance in cultivated barley (Hordeum vulgare ssp. vulgare) and wild barley (H. vulgare ssp. spontaneum). In this experiment, 30 barley genotypes were evaluated in pot culture experiment for root traits and in the field for grain yield and drought tolerance, in two consecutive years. The results indicated that the genotypes with high root dry weight, area, volume and length and root to shoot ratio at depth of 0-30 cm had also extensive root system at the depth of 30-60 cm. The root system size increased when the plants were exposed to drought stress, and the level of increase was higher in the deeper soil layer. The wild barley genotypes Hsp06, Hsp74 and Hsp79 had high averages of the root dry weight, area, volume, and length under both normal and water stress conditions. The results of farm experiment indicated that the cultivated barley genotypes mostly had higher yield potential; however, the wild barley genotypes had more yield stability under drought stress environment. The wild barley Hsp71 was identified with both high yield potential and stability under drought stress. Root dry weight and root to shoot ratio were negatively correlated with grain yield under no water stress condition. Under stress condition, root area, length, and volume were positively correlated with yield stability index. Results indicated that the vigorous root system is not necessarily related to higher grain yield in barley; however, higher yield stability under stress environment is highly related to root system extension.