Molecular characterization of 16 accessions of Nyctanthes arbor-tristis L. collected from different eco-geographical regions of north and central India was carried out to assess genetic diversity using RAPD markers. During RAPD analysis, amplification of genomic DNA of 16 accessions by 40 random primers generated 275 scorable bands with an average of 8.59 bands per primer, of which 180 (65.45%) bands were polymorphic and 95 (34.54%) bands were monomorphic. Each primer generated a unique set amplification product ranging in size from 200 to 2200 bp. The number of bands produced per primer ranged from 4 to 13. The PIC value ranged from 0.703 to 0.909 with an average of 0.848. The RAPD based Jaccard's pair-wise genetic similarity coefficient for Nyctanthes accessions ranged from 0.873 to 0.680. The association amongst different accessions was presented in the form of UPGMA generated dendrogram which clearly segregated all the 16 accessions into two major clusters with equal number of genotypes in each cluster. The major clusters ‘A’ and ‘B’ were further divided into two sub-clusters. The results obtained indicated that N. arbor-tristis accessions, collected from diverse, possessed moderate level of genetic diversity and this information can be further used for the improvement of Nyctanthes for various traits through different breeding methods.