For the purpose of determining the origin of horticultural evergreen azalea cultivars, this study was focused on the natural populations of azalea in Kyushu (south main island of Japan). The Kirishima mountains, the volcanic mountain mass in Kyushu, are an important centre of diversity for the Japanese evergreen azaleas. Rhododendron kiusianum Makino grows above 1000m alt., whereas R. kaempferi Planch. is distributed below 600m alt. Putative natural hybrid populations of these two species are found in the intermediate region (1000–600m alt.). These two species have been clearly distinguished by their respective morphological features. Rhododendron kiusianum has small pink-purple flowers and small elliptical leaves, whereas R. kaempferi has larger red-orange flowers with dark blotches and large oblong leaves. Interspecific hybrids show phenotypes within the range of the two species, especially with regard to flower colour and leaf shape. A morphological cline of these characteristics corresponding to altitude has been observed between these two species. PCR-RFLP analysis of chloroplast DNA detected specific bands for the two species in the 16S rDNA region when digested with HhaI restriction enzyme. Populations of interspecific hybrids were composed of individuals that had a banding pattern of either R. kiusianum or R. kaempferi. This indicates that R. kiusianum and R. kaempferi are clearly distinct species. Furthermore, natural hybrid populations consist of individuals that have one of two cpDNA. Some individuals in the populations of R. kiusianum (T-1430 and T-1030) possess the cpDNA pattern of R. kaempferi, which suggests that cytoplasmic introgression has occurred in the populations of R. kiusianum from R. kaempferi.