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Callus induction is a key step in establishing plant regeneration and genetic transformation. In this study, we present a comprehensive large-scale investigation of the callus induction rate (CIR) in peach trees, which revealed significant variability within the peach germplasm. Notably, the late-maturing cultivars exhibited significantly higher levels of CIR. Moreover, cultivars characterized as having high CIR exhibited potential for the development of adventitious roots (ARs) during callus induction, and a positive correlation was observed between CIR and the ability to regenerate ARs. However, long-term subculture callus lost root regeneration capacity due to changes in cellular morphology and starch and flavonoid content. Additionally, PpLBD1 was identified as a good candidate gene involved in the regulation of callus adventitious rooting in peach trees. Our results provide an insight into the mechanisms underlying callus induction and adventitious root development and will be helpful for developing regeneration systems in peach trees.