© 2020, The Society for In Vitro Biology.Echinacea (Echinacea purpurea L.), also known as purple coneflower, is one of the important medicinal plants commonly used for respiratory infections. The aim of this study was to compare different transgenic hairy root lines for growth and secondary metabolite biosynthesis and to select a superior hairy root line for the production of caffeic acid derivatives (CADs). Agrobacterium rhizogenes-mediated root lines showing hairy root phenotypic characteristics were analyzed by the Southern blot analysis and the copy number of the rolC gene determined. Significant differences were found among seven hairy root lines evaluated for the copy number of rolC gene, root growth, total phenolic, and CAD contents. It was concluded that these differences may be due to the position and copy number of rolC gene in the genome. Across the parameters compared, it was found that line 7 was superior to the other transgenic lines. The results show that the selection of superior transgenic lines is an important factor for the efficient production of secondary metabolites in hairy root cultures of Echinacea.