Sesame (Sesamum indicum L.) of the family Pedaliaceae is an important and old oil crop that is cultivated mainly in the tropical and subtropical regions of Asia and Africa for several economic values. Several molecular markers have been employed to study the genetic diversity of this important crop. The study focused on the genetic diversity through polymorphism information content (PIC) by the use of Simple Sequence Repeat markers among randomly collected 22 Sesame germplasm across 2 ecological zones stored in NACGRAB seed Genebank, Nigeria. The extraction procedure followed Cetyltrimethylammonium Bromide (CTAB) and the recovered DNA were good with average concentration of 337.00 ng/µL and average quality of 1.795. 30 primers were designed but only 12 with highest genome coverage were used to analyzed the genetic data with NTSYS pc ver.2.02 and Power Marker ver.3.5. The PIC ranged between 0.36 in Primer OTO2 and 0.76 in Primer OTO5. The evolutionary relationship was constructed based on the polymorphic primers and according to their ecological locations. The populations were divided to 2 major clads. Of the 2 ecological zones, the more diverse ecological zone is the derived Savana with 13 accessions, while the less diverse is the humid forest with 9 accessions.