Study was executed to differentiate and discriminate (Medicago sativa andTrifolium alexandrinum) using DNA barcoding genes [rbcl]and Cox1 genes. Identification of (Medicago sativa (Rammah 1) was completed via rbcl and Cox1 genesand was identified as Medicago sativa voucher G00199095 ribulose1,5 bisphosphate carboxylase / oxygenase large subunit gene, partial cds; chloroplast Sequence ID: KJ204375.1 or Medicago sativa voucher Ahrendsen 23 for rbcl and Cox1 genes. Identity estimation were listed with 90% as alfalfa, Rammah 1 Genotype ribulose1 /5 bisphosphate carboxylase / oxygenase large subunit gene sequences ID: KJ206375.1] also, identity values of 91.24% were recorded with for alfalfa Rammah 1 Genotype, cytochrome c oxidase subunit I gene cox 1 sequences ID: KJ 204375.1). Trifolium alexandrinum Helaly genotype was identified as Trifolium alexandrinum (Sequence ID: HM850407.1) and Trifolium alexandrinum voucher K-016Hv (Sequence ID: KU234213.1) by rbcl and Cox1 genes respectively. Affiliation of genetic source was revealed for Trifolium alexandrinum with 100 % match with origin which indicate rising possibility for applying discrimination through comparing with Medicago sativa which reflect the lowest genetic likeness with the source. Moreover, we might detect from the available data that we can use DNA Bar-coding Technique in Discriminating the local Egyptian Clover Genotypes and Protect Them internationally. Also, DNA Bar-coding can be used to determine the genetic polymorphism in identifying superior genotypes as source of parental genotypes in Egyptian clover breeding program in future.