Characterization of genetic resources held in both in situ and ex situ GenBanks have important implications for future applications in association mapping studies, genetic selection, breeding, and conservation efforts. This study aimed to evaluate the genetic diversity, population structure, and relationships among 384 Ethiopian barley genotypes collected from various barley-growing regions of Ethiopia using 49 simple sequence repeat (SSR) markers. Analysis of these markers revealed a total of 478 alleles, with an average of 9.755 alleles per locus, of which 97.07% were found to be polymorphic. The Nei's genetic diversity index (h) was 0.654, and the Shannon diversity index (I) was 0.647, indicating moderate-high genetic diversity among the studied barley genotypes. At the population level, the mean percentage of polymorphic loci (PPL) was 98.37%, with h = 0.388 and I = 0.568. The highest genetic diversity was observed in the Arsi population (PPL = 100%, h = 0.439, I = 0.624), while the lowest was in the population from Jimma (PPL = 75.51%, h = 0.291, I = 0.430). Analysis of molecular variance (AMOVA) demonstrated significant genetic differentiation within and between populations (P < 0.001), with 84.21% and 15.79% of the variation occurring within and among populations, respectively. Additionally, the study revealed a coefficient of gene differentiation of 0.053 and a gene flow value of 4.467 among populations. The 384 barley genotypes were grouped into seven genetic clusters based on STRUCTURE, neighbour-joining tree, and principal coordinate analysis, which were significantly correlated with their geographic distribution. These results highlight significant variations within and among populations, providing valuable insights for the design of conservation strategies, including both in situ and ex situ conservation approaches.