Calotropis procera is a weed growing around the arid and semi-arid areas of the tropical and subtropical regions. The species has the potential to replace cotton and resolve the high rates of cotton-based products and at the same time reduce the use of synthetic non-biodegradable fibres. It has been used in various medicinal formulations from a long time. There is a need for developing molecular markers to facilitate breeding and genetic improvement of its varieties. Out of 12.6 million raw reads, 84636 reads were obtained after quality check and filtering. 93.7% of alignment rate and 76.31% of core genes were detected in Bowtie and BUSCO assessment, respectively. An average of 5.5 SSRs/Mb was obtained in 72.349 Mb transcriptome. 25 SSR markers were selected for characterization on 30 individuals representing 3 populations. 13 SSR loci were found polymorphic. In the population structure analysis a moderate level of genetic diversity (Na=3.9231, He=0.633) was predicted. The genetic variation among the population was found to be only 7% whereas within the population, it was 93%. The mean Shannon?s information index obtained was 1.1, showing significant diversity richness in the populations. The Dendrogram showed that the population from Bathinda and Ambala are closely related to each other than those from Barnala. The gene flow between Bathinda and Ambala population was found significantly high (6.439). The present set of loci had a high PIC value of 0.506 that makes them very informative to be further used for large scale studies.