Eritrea is considered a center of origin for sorghum, the main cereal crop in terms of area under cultivation and production in the country. There have been very little genetic diversity studies done on the Eritrean sorghum before. To improve this crop, the knowledge of genetic diversity estimation is needed on the available germplasm. The aim of this study was therefore to asses the extent of genetic diversity within and among 98 sorghum genotypes collected from Eritrea alongside 42 regional reference accessions from the International Crop Research Institute for Semi-Arid Tropics (ICRISAT) using a set of 29 Simple Sequence Repeat (SSR) markers. An average of 4.8 alleles per marker was recorded. The mean polymorphism information content (PIC) value for the SSR loci was 0.52. The Analysis of Molecular Variation (AMOVA) revealed that 12% of the variation resulted from the difference among populations, 31% within individual populations and 57% among individual accessions of the sub populations. Neighbor joining phylogeny tree based on genetic similarity coefficient revealed three distinct groups of clustering with the Eritrean populations further sub clustered into three groups. The Eritrean sorghum accessions from Gash Barka and South regions and South Sudan accessions recorded the highest private alleles. The results of Principal Coordinate Analysis (PCoA) also classified the sorghum accessions into three major groups. Genetic distance matrix revealed that the Eritrean accessions are more related to each other compared to the regional accessions. The existence of higher level of allelic richness, close genetic distance and an isolated clustering of the Eritrean population indicates that the accessions have not been introgressed with foreign genes and are valuable resource for future breeding programs of this crop.