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Roots provide anchorage and enable the absorption of water and micronutrients from the soil for plants. Besides these essential functions, roots are increasingly being recognized as an important organ for the production of diverse secondary metabolites. The goal of this study was to investigate the chemical composition and function of terpenoid secondary metabolites in roots of different cultivars of the popular ornamental plant Chrysanthemum morifolium Ramat. Although C. morifolium is known for rich production of secondary metabolites in its flower heads and leaves, the diversity of secondary metabolites in roots remains poorly characterized. In this study, 12 cultivars of C. morifolium were selected for comparative analysis. From their roots, a total of 20 terpenoids were detected, including four monoterpenes, 15 sesquiterpenes, and one diterpene. The cultivar 'She Yang Hong Xin Ju' exhibited the highest concentration of total terpenoids at approximately 730 µg·g−1 fresh weight. Most cultivars contained sesquiterpenes as the predominant terpenoids. Of them, (E)-β-farnesene was detected in all cultivars. Based on their terpenoid composition, the 12 cultivars were planed into four groups. To gain insights into the function of root secondary metabolites, we performed bioassays to assess their effects on growth of three species of pathogenic fungi: Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahliae. Significant variability in antifungal activity of the root extracts among different cultivars were observed. The cultivar 'Xiao Huang Ju' was the only cultivar that had significant inhibitory effects on all three species of fungi. Our study reveals the diversity of terpenoids in roots of C. morifolium and their function as a chemical defense against fungi. [ABSTRACT FROM AUTHOR]