Cultivars in the species Rosa hybrida L. can be classified into five septet groups according to their karyotypes. The objective of this study was to confirm the measurement of the septets using random amplified polymorphic DNA (RAPD) in various rose cultivars. Three species were comprised of four accessions, and 55 cultivars were subdivided into 37 standard and 18 miniature cultivars. The characteristics of the individual septets in three species were as follows: Rosa multiflora (WD1 and WD2) and R. indica (WD4) belonged to septet A and AA, respectively, and R. rugosa (WD3) belonged to septet C. Moreover, 55 rose cultivars were used in order to assess the contribution of the septets. Nineteen RAPD primers were used for an analysis. As a result of the dendrogram obtained from a cluster analysis, three different rose species were clearly allocated in accordance with their septets. Two accessions of R. multiflora (WD1 and WD2) were grouped together. In this study, the septet concept applied to identify the genetic variations of rose cultivars in more detail. Consequently, RAPD was a reasonable tool for the analysis of genetic backgrounds in rose cultivars.