Cold stress limits alfalfa (Medicago sativa L.) production, yet the physiological and transcriptional differences associated with contrasting cultivar tolerance remain incompletely understood. Here, a cold-sensitive cultivar (CS) and a cold-tolerant cultivar (CT) were compared using physiological, biochemical, and transcriptomic analyses. Following cold acclimation, CT exhibited a significantly higher freezing survival rate than CS (91.67% vs. 4.17%, p < 0.05) and significantly lower electrolyte leakage (p < 0.05). After 7 d at 4 °C, CT had significantly lower malondialdehyde content and higher proline content, antioxidant enzyme activities, chlorophyll content, and PSII photochemical efficiency than CS (p < 0.05), together with weaker ROS-associated staining. RNA-seq identified 711 and 8763 differentially expressed genes in CS after 2 and 26 h of cold treatment, respectively, compared with 663 and 12,009 in CT. These genes were associated with photosynthesis, carbohydrate and energy metabolism, antioxidant processes, membrane lipid metabolism, hormone signaling, and transcriptional regulation. Cultivar-by-time interaction analysis identified 188 and 226 genes whose expression changes differed significantly between cultivars at 2 and 26 h, respectively (adjusted p < 0.05). These findings reveal cultivar-dependent transcriptional trajectories during cold exposure alongside contrasting physiological responses and provide candidate genes for investigating cold tolerance in alfalfa.