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Drought is one of the main environmental factors affecting growth and yield of sorghum in arid and semi-arid areas of the world. In vitro selection of thirty Sorghum bicolor accessions for drought tolerance was undertaken by the use of shoot and root length variations under polyethylene glycol (PEG) stress. Data were recorded at two PEG-6000 levels (0 and -0.7Mpa) on shoot length (SL) and root length (RL). Sorghum accessions WadAkar, Gishish, E315, F.508, F.509, F.511 and KU439 showed low reductions on shoot and considerable increase on root length under PEG stress. A set of 10 EST-SSRs primers related to drought stress was used to assess this trait among sorghum accessions. Six out of the 10 EST were polymorphic (60%). The marker Dsenhsbm99, which code for drought-induced hydrophobic protein, has the highest abundance among the PEG-tolerant accessions. The genetic similarity (GS) for pairs of sorghum accessions was calculated using Jaccard's coefficients. The genetic similarity among these accessions ranged from 0.26 to 0.88. Similarity coefficient matrices based on the data of EST-SSRs, were used to construct a dendrogram. The results obtained from the analysis of the EST markers were in accordance with the results of osmotic stress experiment using PEG. These results may help in breeding more drought[1]tolerant sorghum accessions in the near future