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Background: The classification of diversity in germplasm collections is important for plant breeding. The repetitive element palindromic-polymerase chain reaction (rep-PCR) technique was used to investigate inter-specific diversity within 17 species from the Euphorbiaceae family using REP and BOX primers. Results: The agglomerative cluster analysis was used to evaluate the scoring data. BOX and REP gave amplification with polymorphism of 98.84% and 100% respectively. REP marker demarcated between the subgenus peltatae. Both markers confirmed Jatropha tanjorensis as a natural hybrid between Jatropha gossypifolia and Jatropha curcas. Five random sequences from the rep-PCR gels were chosen, cloned and sequenced. The blast results demonstrated that the amplified products were from the mitochondrial genomes. Conclusion: The rep-PCR molecular tool can be used to characterize diversity in plants as they are suitable for distinguishing eukaryotic genomes effectively.