Chilling requirement (CR) is a key determinant for bud dormancy release in peach [Prunus persica (L.) Batsch]. To examine the genetic basis of CR and facilitate the breeding of climate-resilient varieties, we conducted a genome-wide association study (GWAS) on a diverse panel of 213 peach accessions with their CR phenotypes. The CR phenotypic data collected over 3 years demonstrated high heritability (H2 = 0.86), indicating a strong genetic component. The GWAS analysis identified 52 SNPs associated with CR traits, with major loci clustered on chromosome 1 (17.3–21.2 and 43.7–47.3 Mb) and chromosome 2 (5.2–13.9 Mb), thereby both confirming established loci in the DAM cluster and identifying novel genetic regions. By focusing on regions exhibiting stable CR associations across years and which could be successfully validated by Kompetitive Allele Specific PCR (KASP) assays, 13 candidate CR-related genes were identified. Two highly robust KASP markers derived from loci in chromosome 1 were developed and validated. These markers effectively discriminated between low (<400 h) and high (≥900 h) CR phenotypes. The combined use of these two markers achieved 95.5% accuracy in identifying extreme low-CR phenotypes (CR < 300 h) in peach accessions. The identification of genes linked to these robust markers of CR-related loci and the analysis of their expression during dormancy identified three potentially related with CR trait modulation: a receptor-like protein kinase, a protein kinase and a BED-type zinc finger domain-containing protein. This study provides useful molecular tools for marker-assisted breeding for low-CR peaches and new insights into the complex regulatory network of CR.

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