Apocynum spp., known as Chinese Luobuma species, are perennial herbaceous plants that not only have good ecological characteristics, such as drought resistance, salt resistance, freezing resistance, high-temperature resistance and wind sand resistance, but also have good medicinal and textile value. However, studies on the genetic variation in Chinese Luobuma are rare. In this study, the genotypic variation in the agronomic traits and molecular markers among eight germplasm accessions (referred to as genotypes) of Apocynum spp. was investigated. The accessions were evaluated at two locations in China, Altay and Yuzhong, during a three-year period. Analysis of the variance in yield-related traits revealed significant genotypic variation (p < 0.05) among the eight genotypes at the early flowering and full flowering stages. There were also significant (p < 0.05) genotype × year and genotype × location × year interactions for all the traits except leaf dry weight. In comparison to those evaluated at Yuzhong, the plant height, number of branches, leaf dry weight and stem dry weight at the early flowering stage were greater in Altay, with averages of 991.0 mm, 5.52, 26.41 g and 25.35 g, respectively. There were significant (p < 0.05) differences among genotypes in terms of the quality traits measured at the early and full flowering stages. The crude protein and crude fat content for each genotype at different locations at the early flowering stage in different years ranged from 8.64 to 10.07%. The average flavone (FLA) content was 2.31 mg/100 g. Principal component analysis (PCA) revealed that the G1 genotype in Altay had a higher neutral detergent fiber content and leaf dry weight, and the G2 genotype had a larger stem thickness, branch number and stem-to-leaf ratio. Five DNA sequences, ITS, matK, psbA-trnH, rbcL and trnL-F, were selected for analysis of the molecular variance in Chinese Luobuma. Analyses of molecular variance (AMOVA) based on the nuclear DNA sequences and chloroplast DNA sequences showed that most of the variation occurred within species. Our study indicated the significant genetic variation in Chinese Luobuma for future cultivar domestication. Genotypes with high leaf dry weights and many branches are beneficial for tea production, while tall plants with long internode lengths are valuable for the production of hemp.