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Maytenus emarginata (Willd.) Ding Hou (family Celastraceae), an evergreen and multipurpose tree species of Indian Thar Desert, is a promising source of anticancer agents i.e. sesquiterpene pyridine alkaloid Emarginatine B and Emarginatine F. The knowledge of the genetic diversity of M. emarginata is important for the selection of appropriate genotype with medicinal interest. However, the genetic analysis or breeding system of M. emarginata remains unrevealed, therefore, it is difficult to draw any conclusion regarding its natural genetic diversity, gene flow or genetic differentiation. In the present investigation, start codon targeted polymorphism technique was used first time to examine the genetic diversity of M. emarginata. A total of 51 individuals comprising seven populations, collected from the larger geographical area of arid, semiarid and sub humid regions of western Rajasthan, India, were tested for existing natural genetic diversity in M. emarginata. Of 36 primers screened, 12 SCoT markers produced a total 156 amplicons ranging from 8 to 17 per primer, of which 114 (73%) were polymorphic. Average polymorphic information content, Nei’s gene diversity (h), Shannon index (I) and Percentage of polymorphic loci (Pp) were 0.517, 0.171, 0.257 and 49.54, respectively. Results revealed high level of genetic differentiation (Gst 0.223) and the gene flow v (Nm 1.74) in M. emarginata populations. Analysis of molecular variance showed that percent molecular variance was higher within population (84%) than among population (16%). The cluster analysis carried out by unweighted pair-group with arithmetic mean and the principal coordinate analysis showed high genetic variation among the genotypes and clustered all 51 genotypes into four major clusters while two genotypes in separate groups. High degree of genetic diversity in genotypes/populations may be useful in conservation program and selection of appropriate genotypes for exploitation of this important tree of Indian Thar desert.