Inter simple sequence repeat markers were used to assess the genetic diversity and population genetic structure in 12 populations of Nothapodytes nimmoniana from Western Ghats of India. A total of 16 selected primers produced 103 discernible bands, with 76 (73.7%) being polymorphic. The Nei's gene diversity (h) ranged from 0.1166 to 0.2124, with an average of 0.1518 at the population level and 0.2965 at the species level indicating high genetic diversity. The Shannon's index (I) was estimated to be 0.2189 within populations (range 0.1703–0.2947) and 0.4352 at the species level. The analysis of molecular variance showed that the genetic variation was found mainly within populations (73%), but variance among populations was only 27% and its value, ΦPT = 0.271, P < 0.001, implied that high genetic differentiation among populations. In addition, Nei's differentiation coefficient (GST) was found to be high (0.4882) and the gene flow (Nm) was low (0.5242), confirming the high population genetic differentiation. The unweighted pair-group method using arithmetic average clustering elicited similar results. Based on this, we propose conservation strategy for this plant species.