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Low temperature is one of the most severe abiotic stress factors that limit chrysanthemum growth and development. Natural temperature changes are more complex, and cold stress from a laboratory incubator cannot accurately represent the natural temperature stress. Here, nine separate high-throughput mRNA sequencing technology (RNA-Seq) libraries were generated from the RNA sample of roots from different temperatures, including chilling (Ch), freezing (Fr), and control (CK). The 7 069 and 3 952 differentially transcribed genes were identified as CK vs. Ch and CK vs. Fr, respectively. The Kyoto encyclopedia of genes and genomes pathway (KEGG) enrichment analysis showed that significantly different flavonoid biosynthesis and linolenic acid pathways commonly appeared in CK vs. Ch and CK vs. Fr. Arginine and proline metabolism, lipid metabolism, fatty acid degradation, and fructose and mannose metabolism pathways were found in CK vs. Ch, and only in the CK vs. Fr enrichment metabolic pathway included steroid biosynthesis and monoterpenoid biosynthesis. The transcription of genes on differential metabolic pathways and MYBs were successfully validated using quantitative real-time PCR. At the same time, the antioxidant activity, malondialdehyde, and proline content were analyzed under low temperature. These datasets may aid in understanding and carrying out future studies on the molecular basis of cold stress and contribute to chrysanthemum breeding. [ABSTRACT FROM AUTHOR]