Abstract: The incorporation of rural landscape plants plays a pivotal role in the development and establishment of ecotourism. By studying the genetic diversity of plants in rural landscapes, the richness of genetic variation and the stability of the genetic structure within a population can be revealed. This serves as an important theoretical foundation for the construction of rural ecological landscapes and biodiversity maintenance and is of great significance for the construction of beautiful villages. In this study, eight native landscape species were selected from two ecotourism villages (Changkou Village, Sanming City, Fujian Province; and Paifang Community, Nanjing City, Jiangsu Province). Native rural landscape plant samples were collected from the whole village area. Phenotypic characteristics were measured, and ISSR-PCR experiments were performed. Through analysis of phenotypic features and detection of molecular markers, the Shannon index and Nei’s genetic diversity indexes were calculated to elucidate the levels of genetic diversity of native landscape plant species in different areas. Cluster analysis using phenotypic features identified five types of Liquidambar formosana, six types of Cyclobalanopsis chungii, four types of Quercus glauca, sixteen types of Zelkova serrata, seven types of Toona sinensis, ten types of Aster indicus, five types of Chrysanthemum indicum, and six types of Rubus hirsutus. The phenotypic coefficient variation and Shannon index of the eight native landscape plant species ranged from 0.23 to 0.58, and from 1.51 to 6.74, respectively. In the total area, artificial area and natural area, the Nei’s genetic diversity indexes of the eight native landscape plant species ranged from 0.240 to 0.536, 0.244 to 0.540, and 0.193 to 0.367, respectively. For the eight native landscape plant species, the percentage of polymorphic loci varied from 45.00% to 100.00%, the number of alleles varied from 1.45 to 2.00, and the number of effective alleles varied from 1.30 to 1.64. The results revealed that the phenotypic (Shannon index) and molecular (Nei’s genetic diversity index) genetic diversity levels of the eight native landscape plant species were higher than the average diversity level in numerous other landscape plant species. Additionally, the rural landscape plant species exhibited abundant genetic variation. The genetic diversity of certain rural landscape plant species exhibited a notable degree of variability; however, there were significant differences in the levels of genetic diversity observed between natural and artificial areas. In the context of rural landscape construction, it is important to prioritize the assessment of genetic diversity in rural landscape plant populations. Appropriate measures should be implemented to enhance the even distribution of genetic polymorphisms within the population and preserve the genetic diversity of native landscape plant species. This approach is essential to ensure the long-term stability of rural ecological landscapes.