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Information on the genetic diversity and population structure is essential for developing conservational management programs, especially for threatened species. Decalepis salicifolia (Bedd. ex Hook.f.) Venter is a steno-endemic and critically endangered species of the south Western Ghats of India. The present study used ISSR markers as well as essential oil profiling to reveal the extent and distribution of genetic as well as the chemical diversity of all the twelve known populations of D. salicifolia. A total of 84 amplicons generated using 17 ISSR primers represented an overall 72.34% polymorphism. The highest percentage of polymorphic loci was recorded in the population of Theemalai (40.48%) and lowest in Kokanmalai (4.76%) with an average of 20.04% across all the studied populations. At the species level, the Nei's genetic diversity observed was 0.255 ± 0.186, while Shannon's information index observed was 0.385 ± 0.260. The genetic similarity-based unweighted pair-group method with arithmetic average dendrogram grouped the populations according to their geographic locations, which was corroborated by principal component analysis and Bayesian clustering. Distribution of genetic variance through analysis of molecular variance indicated that 38% variance resides within the population, and 62% variance resides among the populations (P < 0.001). Gas chromatography analyses of root volatiles showed significant variation in the percent content of 2-hydroxy-4-methoxybenzaldehyde. The Mantel test analyses showed a positive correlation between the genetic versus geographic distances. Based on the results, both ex situ and in situ conservation strategies are suggested to maximally preserve the genetic resources of this endangered species. [ABSTRACT FROM AUTHOR]