Echinacea purpurea L. is one of the important ornamental and medicinal plant species. Ploidy manipulation is a valuable tool for improving plant quality or production in E. purpurea as well as in many other plants. To study the segregation of pure ploidy plantlets from colchicine-induced ploidy chimeras in E. purpurea, we used a chimera plantlet that consisted of 1.93% diploid, 35.04% tetraploid, and 63.03% octoploid cells as the source material for experiments. The results showed that three factors significantly influenced the segregation, i.e., the component ratios of different ploidy cells in the chimera, the number of sequential passages, and the methods of segregation culture of the chimera plantlets. Other factors, such as explant types (i.e., leaf, petiole, or root) and 6-benzyladenine (BA) concentrations (i.e., 0.2, 0.4, 0.8, and 1.2 mg·L−1) occasionally influenced the segregation. Pure chromosome-doubled polyploids are not easily obtained in various plant species, so segregation culture of ploidy chimeras may potentially be more effective. The morphological characteristic and content of cichoric acid were compared among diploid, tetraploid, and octoploid plants. Results indicated that tetraploid and octoploid plants had more stunted growth, larger stomata, lower stomata frequency, more chloroplast number in guard cells, and higher cichoric acid content than original diploid lines.