Abstract: Sorghum (Sorghum bicolor L.) is believed to be domesticated for the first time within the geographical range of Sudan, so, studying the genetic variation of sorghum genotypes collections attracts special interest. In this study, 17 sorghum accessions with important agronomic traits, representing four states in Sudan were assayed for polymorphism using Random Amplified Polymorphic DNA technique (RAPD). Ten primers out of 40 tested (A-1, B-20, C-20, D-18, OPE-04, UBC-101, UBC-103, UBC-127, UBC-155 and UBC-157) showed high polymorphism among the accessions. The results indicated 110 polymorphic bands out of 145 bands with percentage of polymorphic bands of 77.6%. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) result showed two major clusters, with a clear trend of grouping of each region. A cluster contained all accessions from eastern Sudan (Red Sea). However, accessions from north Sudan (River Nile), western Sudan (North Kordofan) and south Sudan (Blue Nile) were diverse in another cluster. Based on the results of this study RAPD technique proved to be useful to study genetic variation among the Sudanese sorghum accessions.