Fenugreek (Trigonella foenum-graecum L.) as an important aromatic and medicinal plant is a rich source of the Diosgenin and Trigonelline. Genetic diversity analysis using reliable methods provides useful information for the crop breeding programs and conservation of genetic resource. In the present study, 40 Fenugreek genotypes were evaluated for genotypic diversity using two gene-targeted markers. Seven SCoT and ten CBDP primers amplified 78 and 93 fragments in which 65 and 78 were polymorphic, respectively. The average of polymorphism information content (PIC) for SCoT and CBDP Primers were 0.37 and 0.29 respectively, that showed a higher resolving power of SCoTs than CBDPs. The dendrogram generated using the UPGMA algorithm based on Dice distance coefficient, classified all 40 investigated samples into four main groups. Furthermore, these results were confirmed by principal coordinate analysis (PCoA). Analysis of molecular variance (AMOVA) showed a higher distribution of genetic diversity within populations (66%), than between them (34%). Among all five investigated populations, the highest values of diversity indexes such as number of effective alleles (1.54), Shannon’s index (0.46) and Nei’s gene diversity (0.31) were estimated for population IV indicating the higher variation within this population than others. These results showed a high amounts of genetic variation among tested genotypes, which can be used in breeding programs. Moreover, the results revealed that these two gene-targeted markers are suitable and reliable techniques for DNA fingerprinting and genetic diversity investigations.