Genomic DNA polymorphism and variation in biologically active components of Moringa oleifera were investigated by two different techniques: RAPD-PCR and HPLC analysis. The concentrations of phenolic compounds (cinnamic, caffeic, ferulic, and coumaric acids) and the content of flavonoids (rutin) were quantified by HPLC analysis. Among 20 RAPD primers, 13 were selected to generate polymorphic amplicons producing an average of 5028 bands, of which 83.7% were found to be polymorphic among 57 accessions of M. oleifera (MO 1 to MO 57) and one outgroup (ACB 58) from Banasthali region, India. In total, 57 accessions were clustered into five major groups within the dendrogram. The results of this analysis were further confirmed by principal coordinate analysis (PCoA). There was also high diversity in the concentration of active compounds in the collected samples as revealed by HPLC analysis. The data revealed that the content of polyphenolic compounds varied between 0.06 (sample KVKB) and 210.5 mg/kg (sample BG). The results suggest that there is a strong correlation between phytochemical variables and DNA polymorphism. The study concludes that the results of the genetic, morphological, and phytochemical diversity could be used to select the best accessions of M. oleifera for agricultural cultivation and breeding.