Honeysuckle azalea (Rhododendron luteum) has only a single population occurrence in Greece, on Lesvos Island of the north-eastern Aegean Sea. The genetic diversity of this population was studied in a population of n = 37 individuals randomly sampled in a transect spanning between the highest and lowest natural altitudinal distribution limits in Mt. Ordymnos, SW Lesvos. A modified DNA extraction and isolation protocol was used to overcome problems of DNA quality due to secondary metabolite activity. Genetic variation was investigated based on molecular Inter Simple Sequence Repeat (ISSR) markers. Results showed the presence of a sufficient amount of genetic diversity for the maintenance of adaptive potential. Genetic diversity was lower but comparable to that of other Rhododendron species sampled from the centre of their natural distribution, despite the relatively small population size, negative anthropogenic pressure and population isolation due to the island environment. Some structuring of genetic diversity was indicated based on a PCoA analysis and the genetic distance dendrogram, while spatial autocorrelation was highly significant. Results point towards the need to assign a protection status to the whole area of the species’ natural distribution on Lesvos Island. Moreover, it is proposed that an in situ Gene Conservation Unit (GCU) be established in the core of this population in Lesvos as part of the Network of the European Forest Genetic Resources Programme, while the establishment of ex situ conservation is also advised.