Ethiopia is recognized as a center of diversity for barley, and its landraces are known for the distinct genetic features compared to other barley collections. The genetic diversity of Ethiopian barley likely results from the highly diverse topography, altitude, climate conditions, soil types, and farming systems. To get detailed information on the genetic diversity a panel of 260 accessions, comprising 239 landraces and 21 barley breeding lines, obtained from the Ethiopian biodiversity institute (EBI) and the national barley improvement program, respectively were studied for their genetic diversity using the 50k iSelect single nucleotide polymorphism (SNP) array. A total of 983 highly informative SNP markers were used for structure and diversity analysis. Three genetically distinct clusters were obtained from the structure analysis comprising 80, 71, and 109 accessions, respectively. Analysis of molecular variance (AMOVA) revealed the presence of higher genetic variation (89%) within the clusters than between the clusters (11%), with moderate genetic differentiation (PhiPT = 0.11) and five accessions were detected as first-generation migrants using Monte Carlo resampling methods. The Mantel test revealed that the genetic distance between accessions is poorly associated with their geographical distance. Despite the observed weak correlation between geographic distance and genetic differentiation, for some regions like Gonder, Jimma, Gamo-Gofa, Shewa, and Welo, more than 50% of the landraces derived from these regions are assigned to one of the three clusters.