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The main goal of this study was to improve pure lines for low input agricultural areas of the semi-arid region of Iran and similar environmental conditions. Forty barley pure lines provided from ICARDA along with three check cultivars were evaluated under rain-fed conditions in a semi-arid region of Iran during two years (2011-2013). The experiments were conducted in a randomized complete block design with four replications to estimate genetic variation and heritability for agro-morphological traits. The highest values of phenotypic and genotypic coefficients of variation were observed for the number of grains per spike followed by peduncle length, early vigor and grain yield. The broad heritability estimates ranged from 24% for grain yield to 96% for the number of grains per spike. The highest and lowest expected genetic advance, observed for days to physiological maturity and the number of grains per spike, respectively. Results of stepwise regression and path analysis showed that hectoliter grain weight and thousand grain weight exhibited the highest direct effects on grain yield, respectively. Comparisons between the pure lines and check cultivars indicated that out of 40 pure lines, 27 lines had a higher grain yield. Also, the pure lines No. 29, 13, 9 and 33 were identified as the superior lines for semi-arid environmental conditions. Our results indicate that check cultivars could be improved by selecting for pure lines with taller peduncle and the number of grains per spike, but with heavier grains. Therefore, these lines can be used as genetic material to broaden the genetic basis of barley breeding programs all over the world.