Comprehensive studies on the genetic diversity and structure of endangered species are urgently needed to promote effective conservation and management activities. The big tree rhododendron, Rhododendron protistum var. giganteum, is a highly endangered species with only two known endemic populations in a small area in the southern part of Yunnan Province in China. Unfortunately, limited information is available regarding the population genetics of this species. Therefore, we conducted amplified fragment length polymorphism (AFLP) analysis to characterize the genetic diversity and variation of this species within and between remaining populations. Twelve primer combinations of AFLP produced 447 unambiguous and repetitious bands. Among these bands, 298 (66.67 %) were polymorphic. We found high genetic diversity at the species level (percentage of polymorphic loci = 66.67 %, h = 0.240, I = 0.358) and lowgenetic differentiation (G(st) = 0.110) between the two populations. Gene flow between populations (N-m) was relatively high at 4.065. Analysis of molecular variance results revealed that 22 % of the genetic variation was partitioned between populations and 78 % of the genetic variation was within populations. The presence of moderate to high genetic diversity and low genetic differentiation in the two populations can be explained by life history traits, pollen dispersal and high gene flow (Nm = 4.065). Bayesian structure and principal coordinate analysis revealed that 56 sampled trees were clustered into two groups. Our results suggest that some rare and endangered species are able to maintain high levels of genetic diversity even at small population sizes. These results will assist with the design of conservation and management programmes, such as in situ and ex situ conservation, seed collection for germplasm conservation and reintroduction.