Peach bud differentiation is commercially significant for fruit production. Bud differentiation in peach production is closely linked to chilling requirements. This study investigates the mechanisms of flower bud differentiation in peach varieties L12 and N1 under varying chilling requirements by comparing paraffin sections, hormone content changes, and transcriptomes during four chilling hours and the same physiological stage. At 400 chilling hours, significant changes in flower bud differentiation were observed. During this period, the hormone levels of auxin and gibberellin reached their peak, while abscisic acid levels were at their lowest. This finding indicates that 400 chilling hours has a significant regulatory effect on flower bud development. Transcriptome analysis revealed that 4719 differentially expressed genes were identified in the flower bud differentiation of L12-4 and N1-4, and 2717 differentially expressed genes of L12-8 and N1-2, many of which are involved in IAA, GA, and ABA signal transduction pathways. In N1, the differentially expressed genes AUX/IAA, SAUR, and DELLA were significantly higher than in L12, whereas genes associated with the ABA synthesis pathway, such as PYL2, PYL8, and SRK2A, remained at the lowest level. This study provides a crucial molecular basis for understanding the regulation of plant hormones and their effects on flower bud development under varying chilling hours.