Pyrenacantha volubilis Wight. is a dioecious liana occurring in small, clustered populations. The species had no documented use until the recent past, when it was identified to be a source of a highly traded anti-cancer drug – camptothecin. In the present study, we examine the genetic diversity of 12 fragmented natural populations of P. volubilis using morphological and molecular traits. Twelve polymorphic Inter Simple Sequence Repeat (ISSR) primers and 29 agromorphological traits were used to discriminate the populations using UPGMA and NJ tree algorithms respectively. The ISSR amplicon profile had 133 distinct bands. The maximum number of amplicons were produced by UBC 844 (20 bands) and the average polymorphism was 80.07 per cent. The dendrograms obtained based on molecular and agro-morphological data are in close congruence. The Thiruvananthapuram population stood apart in both the analyses as a discrete outgroup: perhaps a consequence of local adaptation. Substantial genetic diversity exists among populations. This could be tapped in domestication, which is the only way forward for the long-term survival of this species. We also report for the first time a standardized method for extraction of genomic DNA from the leaves of P. volubilis.