Species of the temperate woody bamboo genus Kuruna Attigala, Kathriar. & L.G. Clark (Poaceae: Bambusoideae) distributed in Sri Lanka and southern India, are threatened due to deforestation and habitat fragmentation. The current study focused on the tetraploid woody bamboo Kuruna debilis (Thwaites) Attigala, Kathriar. & L.G. Clark, using twelve variable microsatellite loci to assess the genetic diversity and population structure in six known Sri Lankan populations. Due to the rarity of the species, an exhaustive sampling of accessible plants resulted in a total of only 28 individuals. Nonetheless, the allelic diversity was high at most loci and given the limited distances separating populations (< 65 km apart), they exhibited a fairly high genetic differentiation (FST = 0.113) and strong isolation by distance. Structure, neighbour-joining, and neighbour-net analyses concur in grouping the six K. debilis populations into three genetic clusters consistent with the spatial proximity of the populations: one cluster comprised populations from the Piduruthalagala Mountain and Horton Plains, the second cluster consisted of the population from Adams Peak and the last comprised the populations from the Handapan Ella Plains. Due to multiple indicators of high allelic diversity, the population from the northern Horton Plains (LA124) should be targeted for conservation. Moreover, the population found in Adams Peak (LA159) is also genetically important and critical to the conservation of these species due to its unique genetic diversity. As the first population genetics study of Bambusoideae in Sri Lanka, we anticipate that our results will provide a foundation for future comparative population genetics and conservation studies in the country.