Pterocarpus santalinus L.f. is an endangered tree species that is cherished worldwide for its valuable deep red-colored heartwood, which has multiple uses. This species exhibits a wide range of medicinal properties and offers immense potential for the discovery of bioactive compounds of pharmacological importance. The overharvesting of this species for heartwood has led to a gradual decline in its natural populations, thereby depleting its genetic resources and contributing to the loss of genetic diversity. Therefore, it is essential to assess genetic diversity, as such information allows for the effective conservation and utilization of genetic resources. In the present study, the genetic diversity and population structure among 40 germplasm accessions originating from 8 populations of P. santalinus were analyzed using six cross-species simple sequence repeats (SSR), 20 each of inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. SSR markers revealed higher polymorphism (100 %), followed by ISSRs (82.81 %) and RAPDs (67.50 %), across all the accessions. The mean values of the polymorphic information content (PIC), effective multiplex ratio (EMR), and marker index (MI) were higher for SSRs (0.84, 9.17, and 7.7), followed by ISSRs (0.262, 4.474, and 1.251) and RAPDs (0.204, 3.268 and 0.970, respectively), indicating the superiority of SSRs in detecting polymorphisms in red sanders. Similarly, the estimates of Nei's gene diversity (H) and Shannon's information index (I) varied across the studied accessions, with the highest values for SSRs (H = 0.528, I = 0.923), followed by ISSRs (H = 0.287, I = 0.428) and RAPDs (H = 0.206, I = 0.307). However, the genetic statistic (Gst) or fixation statistic (Fst) and gene flow (Nm) values were higher for RAPDs (0.455 and 0.598) and ISSRs (0.446 and 0.620) than for SSRs (0.383 and 0.489, respectively), indicating significant genetic differentiation of the populations and moderate gene flow among the populations. Analysis of molecular variance (AMOVA) with SSR, ISSR, and RAPD markers revealed that a greater proportion of genetic variation resided within populations (74 %, 69 %, and 66 %, respectively) than among populations (26 %, 31 %, and 34 %, respectively). Hierarchical clustering on the basis of RAPDs grouped most of the accessions according to the population of origin; however, this clustering was different for the ISSR and SSR markers. STRUCTURE analysis divided the accessions into four, two, and three groups with SSR, ISSR, and RAPD markers, respectively, with admixtures detected in all the groups pointing toward shared genetic lineages and diverse genetic ancestry among the accessions of different populations. Overall, the populations of Gumudipoondi of Tamil Nadu and K.V. Bhavi of Andhra Pradesh were genetically more diverse than other populations, as revealed by genetic diversity estimates obtained with SSR and ISSR marker systems; therefore, the accessions of these populations must be prioritized for conservation and utilization in breeding programs for genetic improvement. [Display omitted] • Genetic diversity was assessed among 40 accessions of P. santalinus using 6 cross-species SSR, 20 each of ISSR and RAPD primers. • Cross-species SSRs were more efficient in detecting genetic diversity in the studied populations than ISSRs and RAPDs. • The populations of Gumidipoondi of Tamil Nadu (GTN) and K.V. Bhavi of Andhra Pradesh (KAP) exhibited higher genetic diversity than other populations according to SSR and ISSR marker analysis. • Analysis of molecular variance with SSR, ISSR and RAPD markers showed that genetic variations within populations (74 %, 69 % and 66 %) were greater than among populations (26 %, 31 % and 34 %). • STRUCTURE analysis using SSR, ISSR and RAPD markers divided the populations into four, two and three genetic groups, respectively, with admixtures detected in all genetic groups. [ABSTRACT FROM AUTHOR]