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.