Tef is an indigenous and important food, feed, and cash crop for smallholder Ethiopian farmers. Knowledge of the natural genetic composition of the crop provides the option to further exploit its genetic potential through breeding. However, there are insufficient reports on the genetic variability of Ethiopian tef using a medium-throughput marker system. Hence, the current study was designed to evaluate the genetic variability of released and core germplasm that was collected earlier. Eighty-one tef genotypes collected from eight Ethiopian ecological zones and released varieties were targeted using 14 SSR markers. The study yielded a total of 122 alleles across the entire locus and population. The molecular variance analysis indicated the existence of large genetic differentiation (FIS and FIT = 0.87), with 86% and 13% of the total variation accounted for among genotypes within the population and across all genotypes used for this study, respectively. However, low genetic differentiation among the populations (FST = 0.014, which accounts for 1%) was observed. Multivariate analyses such as clustering and PCoA did not cluster genotypes into distinct groups according to their geographical areas of population. This is presumably due to gene flow among populations. In conclusion, our findings show that there is significant genetic diversity within populations, particularly in the Jimma, Tigray, and released varieties, as well as the presence of private alleles and heterozygosity. The study also indicates the existence of genotypic admixture in the studied materials. The identification of private alleles and their differentiation will be helpful in selecting breeding materials and creating breeding plans.