Cycas nayagarhensis is a recently discovered cycad species in Odisha, India. Only a few populations were located in different regions of Nayagarh district. Due to the small population size, the species is under severe threat. A study was conducted to determine the genetic status of the population using various molecular markers. Inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) were utilised in the study. A total of 20 ISSR markers amplified 132 loci and 10 SSRs amplified 29 loci. The combined data were analysed through STRUCTURE software and revealed seven distinct populations (K = 7). Based on the combined data, the dendrogram segregated the samples into two distinct clusters. The clusters have high bootstrap values and cophenetic correlation, indicating the stability of the groups. A high degree of genetic similarity and less polymorphism showed the alarming conditions of the species. Average similarity among different samples was 0.78, with maximum and minimum similarity being 0.98 and 0.52 respectively. Among the different populations, maximum diversity was observed for Dasapalla (Population 02). Since there are few individuals in these populations, all the populations may be given special attention to conserve the species. This study will help formulate a conservation strategy for the endangered Cycas species.