The endemic and precious timber Pterocarpus santalinus L. f. (Red sanders) is a drought hardy tree species for conservation in peninsular India due to its high risk of illegal timber harvest. It is only found in Eastern Ghats of India, and has become threatened owing to overexploitation of its valuable timber. The development of genomic resources, particularly simple sequence repeat (SSR) markers, is essential for strict implementation of in situ conservation measures and application of DNA information based red sanders genetic resource management. However, a lack of genomic data and e cient molecular markers limit the study of its spatial and temporal population genetic structure, identi cation of diversity hotspots and tree improvement. The current study aims at comprehensive molecular characterization of red sanders and the somatic chromosome counts, ow cytometry and EST-SSR analyses. The results revealed that red sanders is diploid with 2n=20 and the 2C genome size was 0.7872 ± 0.0561pg for the rst time in this species. A total of 3128 EST-SSRs were detected based on 25,854 de novo assembled unigenes from transcriptome data and primer sets designed for 1953 SSRs. Fifty-nine EST-SSR markers were evaluated for polymorphism in the natural populations of red sanders and 13 were found to be suitable for genetic analysis. Two major transcription factor families bHLH and ERF, responsible for abiotic stress and secondary metabolite synthesis were analysed which would provide the foundation for further research on production of medicinally important biocompounds.