Alfalfa （Medicago sativa L.） is a crucial legume forage crop in temperate regions. However， when cultivated in high-latitude regions， chilling injury during spring regrowth stage frequently leads to delayed regeneration and suppressed growth， ultimately reducing both yield and forage quality. To explore the molecular mechanisms of alfalfa to low-temperature stress， we used the cold-tolerant cultivar Longmu 806 as research material. Using a combination of physicochemical analysis and transcriptome sequencing， we analyzed physiological indicators and differentially expressed genes （DEGs） under 4℃ low-temperature treatment over different time periods. A total of 8101 DEGs were identified， and enrichment analysis revealed six metabolic pathways significantly associated with low-temperature stress. Weighted co-expression network analysis （WGCNA） was employed to dissect the molecular mechanisms of alfalfa′s response to low-temperature stress， revealing that MYB-related family genes may play a critical role in alfalfa cold resistance. Enrichment analysis of DEGs in the 'Meblue' module further identified 12 cold-resistance-related DEGs from the tricarboxylic acid （TCA） cycle and glycolysis/gluconeogenesis pathway. This study provides new insights into the cold resistance mechanisms of alfalfa and offers theoretical support for molecular breeding of cold-tolerant alfalfa varieties.