<p>Sorghum [<i>Sorghum bicolor</i> (L.) Moench] is one of the most important multipurpose crops grown throughout the world. It can be used as feed, food, and fiber. Efforts have been made to estimate genetic diversity among Ethiopian sorghum gene bank accession, in situ land race population and sorghum germplasm pools. Although these studies generated useful information, and contribute to knowledge on genetic variability of sorghum in the country, further investigation to unravel the extent of genetic variability and establish the pattern of genetic relationship among sorghum accessions is essential. Therefore, the present study was targeted to explore the degree and patterns of genetic diversity among 96 sorghum genotypes collected from various parts of Ethiopia. The sorghum genotype profiling with 14 simple sequence repeat (SSR) markers resulted 137 alleles with an overall mean of 9.8 alleles per locus. The marker’s polymorphic information content (PIC) and Nei’s genetic distance values ranged from 0.54 to 0.9 and 0.05 to 0.71, respectively. Four major clusters with no strong distinction between geographical origins were obtained by hierarchal clustering using UPGMA analysis. Moreover, analysis of molecular variance (AMOVA) showed 99% variation was observed and attributed to difference within individual accessions, and only a small fraction of the overall variance (1%) was linked to the original collection area. Clustering using UPGMA, PCoA, and STRUCTURE did not sharply cluster the tested sorghum accessions as per their geographical areas of sampling likely due to high gene flow. Among studied populations, those of Amhara, Tigray, and Oromia showed a higher genetic diversity as compared to other populations, and hence, these regions could be considered hot spots for identification of divergent genotypes that can be used in breeding programs. Therefore, the present study generated useful information for genetic improvement and conservation of sorghum.</p>