In this study, some barley genotypes, to which three quantitative character loci (QTLs) were transferred and found as drought tolerant, were used as the research materials and evaluated for their performance in arid conditions in the province of Konya located in the Central Anatolia Region in Turkey. This study was carried out in two separate trials, including in field and controlled conditions. The experiments were laid out in a randomized complete block design (RCBD) with three replications. The parameters, including the leaf relative water content, plant height, spike length, number of spikes per square meter, number of grains per spike, thousand-grain weight, hectoliter weight, harvest index, and grain yield, were investigated. The results of the principal component analysis (PCA) showed that agronomic and physiological measurements explained 84.45% of the first three components for the experiment conducted under field conditions, while 72.89% of the first three components were explained for the controlled experiment. In the controlled trial, where the degree of water stress was more pronounced, the transfer of QTL1 and QTL6 regions into the Bolayır variety and the QTL2 region was transferred to the Baronesse variety resulting into higher grain yield. The Baronesse line (R1), developed by the transfer of the QTL1 region to the Baronesse variety with 819.9 kg da–1 in the field trial, and the Bolayır line (B6), obtained from the transfer of the QTL6 region to Bolayır, with 588.0 kg da–1 in the controlled trial gave the highest grain yields. The lowest grain yields, however, were recorded in the Tadmor variety under the field conditions (418.5 kg da–1) and in the Aydanhanım (A2) line (363.1 kg da–1), in which the QTL2 region was transferred, in the controlled conditions. The developed lines, including Aydanhanım QTL1, Aydanhanım QTL2, Aydanhanım QTL6, Bolayır QTL1, Bolayır QTL2, Bolayır QTL6, Baronesse QTL1, Baronesse QTL2, and Baronesse QTL6 may harbor new alleles associated with drought tolerance traits for breeding drought-tolerant barley cultivars.