Barley production is essential in Egypt. In the present study, 15 different six-rowed Egyptian barley cultivars were studied. To differentiate between the different cultivars under study in terms of morphological characteristics and ISSR, molecular characterization reactions were carried out. Moreover, four cultivars (Giza 123, Giza 126, Giza 136, and Giza 138) were selected for further studies using scanning electron microscopy (SEM). Computational analysis of the DNA barcoding sequences of the two plastid markers rbcL and matK was executed, and the results were deposited in the NCBI database. The morphological traits showed low statistical significance among the different cultivars under study via the data collected from two seasons, suggesting that the mean field performance of these Egyptian cultivars may be equal under these conditions. The results showed that the phylogenetic tree was divided into four groups, one of which contained the most closely related genotypes in the genetic distance, including Giza 124, Giza 130, Giza 138, Giza 136, and Giza 137, which converge in the indicative uses of farmers. The seed coat of the studied cultivars was “rugose”. The elevation folding of the rugose pattern ranged from 11 ± 1.73 µm (Giza 126) to 14.67 ± 2.43 µm (Giza 123), suggesting variation in seed quality and its uses in feed and the food industry. According to the similarity matrix of ISSR analysis, the highest similarity value (93%) was recorded between Giza 133 and Giza 132, as well as between Giza 2000 and Giza 126. On the other hand, the lowest similarity value (80%) was recorded between Giza 130 and (Giza 133 and Giza 132), indicating that these cultivars were distantly related. Polymorphism information content (PIC) ranged from 0.26 for the primer ISSR UBC 835 to 0.37 for the primers ISSR UBC 814 and ISSR UBC 840. The current study showed that the matK gene is more mutable than the rbcL gene among the tested cultivars.