Calotropis procera is emerging as a new, yet undomesticated, resource of fibre comparable to cotton and kapok. Screening of efficient genotypes from its wild populations would be a useful pre-domestication process. The desired genotypes can then be improved through conventional breeding programmes to develop a domesticated variety. Molecular markers play a major role in modern breeding systems. Thus, an efficient marker resource for C. procera would prove useful in germplasm selection during breeding programmes. In this study, we undertook an initial step of Simple sequence repeats (SSR) marker development for C. procera, which could be applied for germplasm selection. Furthermore, using the developed markers, we assessed the genetic diversity base within its wild populations which could be useful to identify the hotspot areas of germplasm collection. Out of 94,636 de novo assembled transcripts, 9148 sequences were found to contain 12,884 SSRs at a density of 5.5 SSRs/Mb. Twelve SSRs were found as polymorphic with a mean polymorphic information content of 0.575. We observed a moderate level of genetic diversity (Na = 3.625, Ho = 0.58) in the studied populations. Mantel's test showed significant correlation between the geographic distance and the genetic distance (r = 0.147, P = 0.010). Sirsa was found as a genetically most diverse population followed by Barnala while Gurdaspur was found with the least genetic diversity. These genetically diverse populations can serve as an important resource for effective germplasm collection for breeding programmes.