Walnuts (Juglans regia L.) are an economically important woody crop, but spring frost poses a serious threat to their growth and productivity. However, the molecular mechanisms underlying walnut responses to freezing stress remain largely unknown. In this study, transcriptome analyses were performed on cold-tolerant and cold-sensitive walnut varieties subjected to freezing stress. A total of 9611 differentially expressed genes (DEGs) responsive to freezing stress were obtained, of which 2853 were common up-regulated and 2880 were common down-regulated in both varieties. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed 15 significantly enriched pathways in both varieties, including flavonoid biosynthesis. A simplified walnut flavonoid biosynthesis pathway was constructed, encompassing 36 DEGs encoding 13 key enzymes, demonstrating that flavonoid biosynthesis in walnut is significantly activated under freezing stress. Furthermore, weighted gene co-expression network analysis (WGCNA) identified a regulatory network centered on the JrCBF genes and uncovered 34 potential interacting genes. Collectively, these findings provide novel insights into the molecular responses of walnut to freezing stress and establish a foundation for elucidating the mechanisms underlying walnut cold tolerance.