AbstractSudan is the birthplace of sorghum, and vast genetic diversity exists among its wild relatives. To assess the genetic potential of Sudan wild sorghum accessions, we used 41 stay-green-specific microsatellite (SSR) markers to analyze the genetic variability and population structure of 256 accessions. Overall, 17 SSR markers were polymorphic, with 55 alleles on average 3.3 per locus. The polymorphic information content (PIC) ranged from 0.49 to 0.57, with an overall mean of 0.53, indicating the potential of these markers for capturing the genetic construction of wild sorghum. Linkage disequilibrium analysis identified the two most informative markers, Xcup05 and Xtxp212. Accordingly, the Nei gene diversity of the populations varied from 0.032 to 0.127, with an overall mean of 0.083. Molecular variance analysis (AMOVA) demonstrated that 99% and 1% of the genetic variations were within and among populations (Fst = 0.066; P 0.001), respectively. However, gene flow (Nm) values varied from 0.058 in populations 1 and 2 to 1.018 in populations 2 and 3. Neighbor-joining trees identified from 21 Sudanese wild sorghum accessions clustered closely to the universally drought-tolerant landrace B35. Structural analysis generated the highest Delta K value (58.2) at K = 2, revealing two distinct subpopulations. While this work provides valuable information about the potential of sorghum wild relatives from Sudan as sources for stay-green drought tolerance, further research should be directed toward identifying the exact mechanisms and genes underlying this stay-green trait using advanced molecular omics techniques. In conclusion, this study highlights the potential role of Sudanese sorghum accessions as reservoirs of ready-to-use stay-green genes for the design of climate-resilient sorghum cultivars in drought-prone areas of Sudan and beyond. However, these wild relatives would require extensive pre-breeding and validation efforts before their genes can be effectively incorporated into elite cultivars.