Chalcone synthase （CHS） genes constitute a highly conserved gene family in plants. Although these genes have been implicated in regulating tobacco growth and development， their potential roles in low-temperature stress and abscisic acid （ABA） biosynthesis remain uncharacterized. This study cloned the ortholog of CHS in tobacco and investigated the role of NtCHS in low-temperature stress and ABA biosynthesis through bioinformatics analysis， expression profiling， stress tolerance assessment in RNAi-silenced lines， and untargeted metabolomics analysis. RT-qPCR results showed that NtCHS was highly expressed in tobacco flowers and leaves， and its expression was strongly induced by both low temperature and ABA. The dual-luciferase assay indicated that the region from -622 to -1312 bp upstream of the NtCHS gene may contain key transcriptional regulatory elements for its low-temperature response. Compared with the wild-type K326， total flavonoid content was significantly decreased in NtCHS RNAi-silenced lines. After 24 h of low-temperature treatment， hydrogen peroxide， superoxide anion and malondialdehyde in the RNAi-silenced lines were markedly elevated， indicating aggravated oxidative damage and ultimately impaired low-temperature tolerance. Untargeted metabolomic analysis indicated that， compared with the wild-type K326， the content of nordihydroguaiaretic acid， an inhibitor of ABA biosynthesis， was significantly upregulated in the NtCHS gene RNAi-silenced lines， whereas the contents of ABA biosynthesis precursors including neoxanthin， farnesyl pyrophosphate and mevalonic acid were significantly downregulated in these lines. Furthermore， expression of ABA biosynthesis genes was significantly downregulated in RNAi-silenced lines， whereas genes involved in ABA catabolism was significantly upregulated. In conclusion， our findings demonstrate that NtCHS is an important regulator of low-temperature tolerance and ABA biosynthesis in tobacco， providing a new target for improving cold tolerance in tobacco varieties.