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Scaffold proteins, which are known as hubs controlling information flow in cells, can function in a diverse array of biological processes in plants. The BTB/TAZ domain-containing scaffold proteins are associated with multiple signaling pathways in plants. However, there have been few studies of the roles of BT scaffold proteins in chrysanthemum to date. In this study, four CmBT genes named as CmBT1 , CmBT1-LIKE1 (CmBT1L1), CmBT1-LIKE2 (CmBT1L2) , and CmBT5 were cloned based our previous RNA-seq database. The four CmBT genes showed distinctive expression patterns both in different tissues and in response to different stimuli, such as light, sugar, nitrate and auxin. Knockdown of the four CmBTs facilitated the development of adventitious roots and root hair in chrysanthemum. Transcriptome sequencing analysis revealed thousands of differentially expressed genes after knockdown of the four CmBT genes. Moreover, functional annotation suggested that CmBTs play a tethering role as scaffold proteins. Our findings reveal that CmBTs can negatively regulate root development of chrysanthemum by mediating nitrate assimilation, amino acid biosynthesis, and auxin and jasmonic acid (JA) signaling pathways. This study provides new insights into the role of CmBTs in root development of chrysanthemum. • Knock-down of CmBTs significantly promotes adventitious roots and root hairs development in chrysanthemum. • CmBTs affect nitrate assimilation and amino acids biosynthesis in roots of chrysanthemum. • Phytohormones are involved in root development under the regulation of CmBTs in chrysanthemum. [ABSTRACT FROM AUTHOR]