One of the main crops with major global economic and agricultural importance is barley (Hordeum vulgare L.). For the purpose of effectively conserving, using, and improving this essential crop, it is imperative to comprehend the genetic diversity and relatedness among barley landraces. Furthermore, the genetic variability of twelve Libyan barley genotypes was assessed based on different morphometric traits and molecular analysis using 7 ISSR and 8 SRAP primers. The results of ISSR primers revealed 55 bands while the SRAP primers generated 46 bands. The primers used in the two approaches revealed that12 unique bands among the 12 barley genotypes. The molecular analysis of ISSR and SRAP revealed 76.36% and 45.65% polymorphisms, respectively. A total of 62.38% of both methods' polymorphism was found. Due to their shared heritage, the 12 barley genotypes under investigation showed a notable degree of genetic diversity, as indicated by the cluster analysis. In the regard, the principal component analysis (PCA) and heat map results validated the cluster analysis constructed on the two molecular techniques. Finally, the ISSR and SRAP patterns' similarities are congruent with the morphological traits of these accessions, as well as their relatedness and geographic distribution.