<p>Sorghum is an important crop in sub-Saharan Africa sustaining many livelihoods. However, despite its importance, sorghum continues to have limited research attention and priority and is categorized as one of the Neglected and Underutilised Species. The paucity of current information on genetic diversity and structure of sorghum accessions being cultivated, improved, introduced, and or conserved across east and southern Africa remains limited. <i>Ex situ</i> genetic diversity and population structure for 177 sorghum accessions from diverse sources was studied using high throughput Diversity Array Technology markers. A total of 15,220 DArTseq SNP markers were generated with over 90% call rate and 95% reproducibility. DArTseq SNP markers had an average polymorphic information content of 0.21 indicating the discriminatory power of DArTSeq markers. Genetic relationships estimated using discriminant analysis of principal components and ancestry coefficients revealed eight populations. Observed heterozygosity was lower than the expected heterozygosity across populations with a mean inbreeding coefficient (F_IS) of 0.82. Analysis of molecular variance found 83% of the variation to be attributed to within-population variance. A high divergence (F_st = 0.16) obtained from AMOVA suggests high genetic differentiation among populations. Genetic diversity indices Shannon-wiener index (H) (3.37–3.95), Stoddard and Taylor index (G) (29–52) and Simpson’s index (λ - Lambda) (0.90–0.99) were high across populations. Nei’s unbiased gene diversity ranged from 0.13 to 0.23. The results suggest a high differentiation among sorghum accessions under study. Implications with respect to insights to guide identification, selection and testing of plant genetic resources for food and agriculture with unique genes and traits to address demand-led crop improvement and market needs, conservation, and deployment in farmer fields are shared.</p>