The present experiment was conducted in Genetic Resources Research Dept., at Bahteem Research Station during 2017/2018 and 2018/2019 seasons, to estimate genetic biodiversity among barley accessions using various methods. The results showed that the flag leaf anthocyanin coloration and awn color in anthocyanin were classified into two groups, present or absent. Also chlorophyll color and type of leaves were classified into two groups, density and wild linear, light green and linear. In addition, growth habit in awns was classified into two groups, erect and semi erect. On the other hand, wax in flag leaf in first awn and spike were classified into three groups, very weak, weak and medium. Moreover, terminal color in anthocyanin was classified into three groups, very weak, medium and strong. As well as, density of awns was classified into three groups, very density, dense and medium. Otherwise, wax of flag leaves was classified into four groups, very weak, medium, strong and very strong. The results in quantity accession no. 38 gave the highest 1000-kernel weight and kernel weight. The correlation coefficient positive significant between awnless and 1000-kernel weight, also 50% heading date and plant height. Molecular markers analysis evaluated using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). A high level of polymorphism was found with both RAPD and ISSR markers. In RAPD analysis total 111 bands out of 59 polymorphic bands and 52 monomorphic, while ISSR analysis total 76 bands out of 37 polymorphic bands and 39 monomorphic bands.