Abstract: Carya illinoinensis is an important woody oil and precious dry fruit tree species. However, low-temperature can limit its growth and affect its yield and quality. By studying the physiological responses and gene expression changes of C. illinoinensis under low-temperature treatment, this research aims to enrich the understanding of its cold-tolerance mechanism and provide a reference for molecular assisted breeding for cold resistance. The annual seedlings of cold tolerant C. illinoinensis from Xinjiang provenance (XJ) and cold sensitive “Shaoxing No.1”C. illinoinensis (SX) were treated. The normal temperature treatment at 25℃ was set as the control group, and the low-temperature treatment group was set at -20℃, with a treatment time of 12 hours. After the treatment, the leaves of C. illinoinensis seedlings were collected to determine the activities of superoxide dismutase (SOD), and the contents of proline and malondialdehyde (MDA), in order to evaluate the physiological changes of XJ and SX C. illinoinensis under low-temperature. Meanwhile, the leaf RNA of the control and low-temperature treated plants was extracted for transcriptome sequencing. Through analyzing the changes in differentially expressed genes of XJ and SX under different treatments, the physiological and molecular mechanisms of the differences in their cold-resistance abilities were preliminarily analyzed. After low-temperature treatment, compared with the control, the physiological indicators in response to low-temperature, SOD, proline, and MDA, increased by 58.11%, 39.30%, and 3.42% respectively in XJ, while in SX, they increased by 152.53%, 94.52%, and 7.97% respectively. The results of transcriptome sequencing showed that low-temperature treatment significantly induced the low-temperature responsive genes in both XJ and SX, and the CBF-like genes were induced to a greater extent in XJ. Genes related to growth and development, photosynthesis, and cell wall modification were significantly inhibited. At the same time, there were gene expression changes caused by genetic differences between the two types of C. illinoinensis. On the one hand, XJ genetically had up-regulated genes related to oligosaccharide biosynthesis and metabolism, while genes related to low-temperature associated cell surface receptors, membrane receptor signal transduction pathways, meristem tissues, and growth and development were more strongly inhibited. Additionally, under low-temperature stress, genes related to brassinosteroid biosynthesis and signal transduction pathways in XJ were significantly enriched in the up-regulated gene set compared to SX. Meanwhile, pathways such as RNA metabolism, modification, and regulation were enriched in the uniquely down-regulated genes of XJ under low-temperature stress. The cold tolerant XJ has a strong oligosaccharide synthesis ability, which enhances its material basis for coping with low-temperature. At the same time, it may also weaken the sensitivity of cells to low-temperature by inhibiting low-temperature related cell surface and membrane receptor genes, thereby reducing the adverse effects of low-temperature on plant physiology and metabolism. Under low-temperature stress, XJ also participates in the low-temperature stress response by enhancing the biosynthesis and signal transduction ability of brassinosteroid and increasing the gene expression of the CBF pathway, thus improving its cold resistance ability.