This study was initiated to reveal genetic relationship of 25 tef (Eragrostis tef (Zucc.) accessions by using 10 SSR markers and to determine DNA content variation by using flow cytometer analysis. Ten markers produced a total of 18 alleles and 11 of those were polymorphic. The mean polymorphism rate was 66.6%. The highest polymorphism information content value was obtained from marker CNLTs370 with 0.69 while markers CNTLs11 and CNTLs133 produced monomorphic bands only. UPGMA analysis divided 25 tef genotypes into three main clades. The accessions PI193511 and PI195934 were distinctly separated from the others. No ploidy differences were determined among the 25 tef accessions. 2C mean nuclear DNA content ranged from 1.406 pg to 1.510 with mean of 1.460 pg. The results of this study indicated that SSR markers successfully determined genetic relationship of 25 tef accession although they had a low rate of polymorphism. This study also revealed that available tef related SSR markers should be optimized before use and their efficiency may vary based on tef genotypes or accessions used.