Genetic variability of seven Egyptian barley cultivars was analyzed utilizing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and random amplified polymorphic DNA (RAPD) techniques.  The results of SDS-PAGE produced 12 bands ranging from 135 to 11 KDa. Two polymorphic bands at molecular weight (MW) 13 and 12 KDs were detected with 16.6% polymorphism among barely cultivars. In the other hand, five RAPD primers were employed to assess the genetic diversity and relationships between the seven Egyptian barley cultivars. RAPD analysis exhibited 93 amplicons (86% polymorphism) with an average number of 18.6 amplicons per primer. Genetic similarity of RAPD and protein input ranged from 0.31 to 0.83. The dendrogram of combined data had clustered all the cultivars into two main clusters; the first one containing all barley cultivars except Giza 123 cultivar which formed a separate cluster indicating that the genetic background of this cultivar was distinct from all cultivars. The results obtained from RAPD and protein analysis exhibit different level of polymorphism. RAPD profile is more suitable technique than SDS-PAGE in assessing genetic variation among the seven barely cultivars. Moreover, the present results suggest that, the increasing number of primers and using more different markers could be more accurate to discriminate the genetic variance between barley cultivars. Subsequently, this could be useful to differentiate between barley cultivars in the breeding program.