Decalepis arayalpathra, a critically endangered plant species, has a restricted and fragmented population in Southern Western Ghats, India. This study is a first attempt to evaluate genetic diversity and population structure in the nine wild populations of D. arayalpathra based on molecular pattern realized through the marker assays. Principal coordinate analysis (PCoA) and Nei's unweighted pair-group method with arithmetic average (UPGMA)-based hierarchical clustering of both the marker assays suggest strong genetic clustering between the individuals corresponding to their geographical ranges. Mantel test also corroborates a close genetic proximity between genetic and geographic data (r = 0.389). Population genetic analysis revealed low levels of gene flow [inter-simple sequence repeat (ISSR) = 0.289 and random amplified polymorphic DNA (RAPD) = 0.847] between the populations, in line with high genetic differentiation (Gst = 0.531 with ISSR and 0.440 with RAPD), which was also supported by analysis of molecular variance (AMOVA), that 54 % (ISSR) and 64 % (RAPD) total variation resided within populations. Bayesian model-based STRUCTURE analysis detected three genetic clusters showing the high degree of admixture within population. Based on the findings, such as inbreeding depression and the loss of genetic diversity, suggestions for conservation strategies are provided to preserve the genetic resources of this endangered species.