Abstract: This study aims to investigate the effects of postharvest hot water treatment (HWT) on chilling injury (CI) and storage quality of peach fruits. 'HuijingMilu' peaches were utilized as the experimental materials. Three hot water treatments (HWT1 (40°C for 20 min), HWT2 (46°C for 10 min), and HWT3 (52°C for 3 min)) were examined on various quality parameters during cold storage at 4°C for 28 days, followed by room temperature storage (25°C) for 1 and 3 days. Specifically, some parameters were measured in the period of storage, including the fruit quality attributes, CI development, and antioxidant responses. The experimental results demonstrated that there were some significant differences between the treatment groups and the untreated control group (CK). Notably, the HWT3 emerged as the most effective treatment in the multiple aspects of fruit quality during preservation. While both HWT1 and HWT2 shared some positive effects. HWT3 treatment has successfully mitigated the development of CI symptoms and the increase in the respiratory rate, particularly for better ethylene evolution. Furthermore, the accumulation of malondialdehyde (MDA) was effectively suppressed to prevent the excessive increase in the permeability of the cell membrane, which was closely associated with cellular membrane integrity. Among them, the MDA was one of the most key indicators of membrane lipid peroxidation. Quantitative analysis revealed that the peach fruits after HWT3 treatment were remarkably reduced by 45.5% in the MDA content at day 14 of storage, compared with the CK. The relative electrical conductivity in the CK exceeded by a factor of 1.3 with the statistical significance (P<0.05) at day 29 of storage, compared with the HWT3 group. The HWT3 treatment was performed better to preserve the important antioxidant compounds within the fruit tissue. The degradation of several key antioxidant components was effectively delayed after HWT3 treatment, including vitamin C, flavonoids, total phenolic compounds, and glutathione. At day 28 of storage, the content of vitamin C in HWT3-treated fruits maintained at levels 2.52 times higher than that in the CK (P<0.05), indicating exceptional preservation of the essential nutrient. The peach fruits after HWT3 treatment exhibited a high resistance to oxidative stress during the middle phase of storage. Specifically, the levels of reactive oxygen species were reduced significantly, such as hydrogen peroxide (H₂O₂) and superoxide anion (O₂^-). Significant upregulation (P<0.05) was also observed in the expression of the key antioxidant defense genes, including the encoding superoxide dismutase (SODs), catalase (CATs), and glutathione S-transferase (GSTs). These molecular changes suggest that the HWT3 treatment enhanced the inherent antioxidant defense of fruits at both the biochemical and genetic levels. In conclusion, the high quality of fruits after HWT3 treatment was achieved to successfully preserve the antioxidant compound levels during late storage, while enhancing the antioxidant gene expression during middle storage. Reactive oxygen species were effectively inhibited to reduce the damage and subsequently CI in peach fruits. An optimal HWT3 treatment was also obtained to maintain the peach fruit quality during cold storage and subsequent shelf life. The findings can greatly contribute to the valuable insights into the molecular and biochemical mechanisms underlying the protective effects of hot water treatment against CI in peach fruits. A scientific basis can also provide for practical application in postharvest fruit preservation.