Background/Objectives: Despite its economic importance, the genome-wide genetic diversity of sesame germplasm conserved in the Ethiopian national ex situ collection, a proposed center of origin, remains inadequately characterized. This study assessed genome-wide genetic diversity and population structure in 188 sesame accessions from six Ethiopian Agricultural Research Centers using DArTSeq-based SNP markers. Methods: After quality filtering, 5163 high-quality markers were retained from the original set of 12,302 SNPs. Mean expected heterozygosity (He = 0.201) exceeded observed heterozygosity (Ho = 0.193), reflecting sesame’s predominantly self-pollinating nature. Results: The SNPs showed a transition/transversion ratio of 1.17:1 and an uneven distribution across 16 linkage groups. STRUCTURE, PCA, DAPC, and neighbor-joining cluster analyses revealed a clear hierarchical population structure with distinct clusters and varying admixture. Accessions from Assosa (AARC) and Bako (BARC) were genetically uniform, whereas Werer (WARC) and Gambella (GaARC) were major diversity reservoirs, exhibiting high heterozygosity and gene diversity. Pairwise FST values ranged from 0.001 to 0.356, and AMOVA indicated that 30–43% of variation occurred among collections and 57–70% within collections, highlighting substantial intra-collection diversity. Conclusions: The findings highlight that specific research centers were identified as key sources of genetic variation for breeding, conservation, and association mapping to enhance the improvement in agronomic and adaptive traits in sesame for the Ethiopian sesame gene pool.