Seventeen promising lines of barley (Hordeum vulgare L.) were grown in two consecutive growing seasons to evaluate the morphological, genetic variation and yield performance under field experimental conditions compared with three varieties ( Giza 2000, Giza 132 and Giza 133). Twenty genotypes were distributed in a randomized complete blocks design in three replications. Twenty nine morphological traits were described using UPOV (The International Union for the Protection of New Varieties of Plant) Guidelines. Results showed that most of the morphological and yield characters were very different among all genotypes. Meanwhile, the heading data and maturity date characters varied among the lines comparing to their check varieties. The seed yield components had the highest mean values in lines 1 to 7, as the seed index had the lowest weight in two lines and reached the superior weight in 10 lines. The molecular characterization was carried out using 15 anchored-ISSR primers. The selected ISSR primers amplified a reliable banding pattern with the studied barley genotypes. The total percentage of polymorphism was 84.5%, recorded for 240 polymorphic bands out of 284 total bands. The fifteen used ISSR primers were able to characterize 14 lines and 3 check varieties with total 35 unique markers. The highest genetic similarity was 0.84, and the lowest similarity index revealed 0.60. The resulted phylogenetic dendrogram clustered the twenty genotypes into two main clusters, where the check varieties grouped together with 0.75 similarity. The most noted observation was that the high level of genetic homogeneity and the high similarity matrix (ranged from 0.64 to 0.84 similarity) recorded for within and between genotypes. Based on the barley yield traits, out of ten revealed Eigen values, the first three PCs assigned 69.67% variation for all studied traits. These results concluded that 17 lines may be considered as genetic resources for indirect and direct selection criteria in breeding program to improve the grain barley quality and quantity.