Evaluation and characterization of genetic resources maintained at both in situ and ex situ GenBanks have important implications for future utilization in association mapping studies, genetic selection, breeding and conservation activities. The main objective of this study was to evaluate the genetic diversity, population structure and relationship of 384 Ethiopian barley genotypes collected from different barley growing regions of Ethiopia using 49 simple sequence repeat (SSR) markers. Analysis of these 49 SSR markers amplified a total of 478 alleles with an average of 9.755 alleles per locus were obtained of which 97.07% of the loci were observed to be polymorphic. Nei’s genetic diversity index (h) was 0.654 and the Shannon diversity index (I) was 0.647, indicating that the genetic diversity in barley genotypes studied was moderately high. At the population level, mean per cent of polymorphic loci (PPL) showed 98.37%, h = 0.388 andI = 0.568. Highest level of genetic diversity was observed in the Arsi population (with PPL = 100%, h = 0.439, I = 0.624); the lowest value observed was in population from Jimma (with PPL = 75.51%, h = 0.291, I = 0.430). Analysis of molecular variance (AMOVA) result showed 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. Further, genetic variation analysis showed a coefficient of gene differentiation of 0.053 and a gene flow value of 4.467 among populations. The 384 barley genotypes were divided into seven genetic clusters according to STRUCTURE, neighbour-joining tree and principal coordinate analysis, correlating significantly with geographic distribution. These results signified presence of significant variations within and among populations (P\0.001), with the highest of the variation occurringwithin populations. The results will also assist with the design of conservation strategies, such as genetic protection via both in situ and ex situ conservation.