Successful production and development of stable and adaptable cultivars only depend on the positive results achieved from the interaction between genotype and environment that consequently has significant effect on breeding strategies. The objectives of this study were to evaluate genotype by environment interactions for grain yield in barley advanced lines and to determine their stability and general adaptability. For these purposes, 18 advanced lines along with two local cultivars were evaluated at five locations (Gachsaran, Lorestan, Ilam, Moghan and Gonbad) during three consecutive years (2012–2015). The results of the AMMI analysis indicated that main effects due to genotype (G), environment (E) and GE interaction as well as four interaction principal component axes were significant, representing differential responses of the lines to the environments and the need for stability analysis. According to AMMI stability parameters, lines G5 and G7 were the most stable lines across environments. Biplot analysis determined two barley mega-environments in Iran. The first mega-environment contained of Ilam and Gonbad locations, where the recommended G13, G19 and G1 produced the highest yields. The second mega-environment comprised of Lorestan, Gachsarn and Moghan locations, where G2, G9, G5 and G7 were the best adapted lines. Our results revealed that lines G5, G7, G9 and G17 are suggested for further inclusion in the breeding program due to its high grain yield, and among them G5 recommended as the most stable lines for variable semi-warm and warm environments. In addition, our results indicated the efficiency of AMMI and GGE biplot techniques for selecting genotypes that are stable, high yielding, and responsive.