<p>Low temperature is an important limiting factor affecting the growth and geographical distribution of sainfoin. The new cold-tolerant P4 (RC) and sensitive 13709 (SC) lines were treated with low temperature (4℃) for 0, 12, 24, 48, and 72&#xa0;h. The physiological and biochemical indexes and gene expression changes were measured to compare the responses of the two lines to low-temperature stress, and to reveal the molecular physiological mechanism of RC in responding to low-temperature stress. High-throughput Illumina HiSeq sequencing was used to analyze the gene expression of sainfoin in response to low temperature stress. Sequencing results showed that 6523 and 5733 differentially expressed genes (DEGs) were identified in RC and SC under low-temperature stress, respectively. GO analysis showed that DEGs are mainly biological processes, involved in abiotic stimulation, compound metabolism, and stress response. KEGG enrichment analysis showed that DEGs were mainly concentrated in plant hormone signaling, plant pathogen interaction, and MAPK (Mitogen-activated protein kinase) signaling pathway. After responding to low temperature stress, the gene WRKY2209 (TRINITY_DN7217_c0_g1) involved in MAPK signaling pathway metabolism and Abscisic acid (ABA), Gibberellin (GA), Brassinosteroid (BR), and Auxin (AUX) genes involved in the metabolism of signals TRINITY_DN3521_c0_g1, TRINITY_DN1974_c0_g1, TRINITY_DN36491_c0_g1, and TRINITY_DN90303_c0_g1 were significantly up-regulated. The MAPK signaling pathway may be involved in the response to low temperature stress in sainfoin. Additionally, plant hormones such as ABA, AUX, BR, and GA also play an important role in the response of sainfoin to low-temperature stress. This study enriched the research of the cold resistance mechanism of sainfoin and provided excellent candidate genes for breeding new varieties.</p> Graphical abstract <p></p>