Sesame (Sesamum indicum L.) plays a crucial role in Ethiopian agriculture, serving both subsistence and commercial purposes. However, our understanding of the extensive genetic diversity and population structure of Ethiopian sesame remains limited. To address this knowledge gap, we genotyped 368 Ethiopian sesame germplasms, categorizing into four distinct breeding groups: Accessions, landraces, improved varieties, and wild types, using a comprehensive set of 28 polymorphic markers, including 23 simple sequence repeat (SSR) and five Insertion-Deletion (InDel) markers. These markers ensured robust genomic representation, with at least two markers per linkage group. Our results unveiled substantial genetic diversity, identifying a total of 535 alleles across all accessions. On average, each locus displayed 8.83 alleles, with observed and expected heterozygosity values of 0.30 and 0.36, respectively. Gene Diversity and Polymorphic Information Content (PIC) were recorded at 0.37 and 0.35. The percentage of polymorphic loci varied significantly among breeding groups, ranging from 8.00% to 82.40%, indicating high diversity in accessions (82.4%), moderate diversity in improved varieties (31.20%) and landraces (29.60%), and limited diversity in wild types (8.00). Analysis of Molecular Variance (AMOVA) results emphasized significant genetic differentiation among populations, with substantial diversity (P < 0.001) within each population. Approximately 8% of the entire genetic diversity could be attributed to distinctions among populations, while the larger proportion of genetic diversity (92%) resided within each individual sesame population, showcasing heightened diversity within each group. Our study's findings received support from both Bayesian clustering and Neighbor-joining (NJ) analysis, reaffirming the credibility of our genetic structure insights. Notably, Population structure analysis at its highest ¿k value (k = 2) revealed the existence of two primary genetic clust