ABSTRACT Accurate measurement of DNA damage is critical for therapeutic optimization in proton‐based targeted radiotherapy. This research pioneered the first investigation into the groove damage distribution mechanism in clustered B‐DNA damage induced by proton radiation. Compared to previous studies, this work introduces methodological innovations by constructing nucleosome models with complete B‐DNA base sequences for the first time, enabling more microscopic and precise simulation of DNA damage. Moreover, the research adopts a novel perspective by incorporating DNA intrinsic characteristics and local functional heterogeneity, specifically elucidating differential damage susceptibility between major and minor grooves to uncover the intrinsic characteristic of DNA damage. Through improvements to the PDB4DNA program and DBSCAN algorithm, we found that: (1) in simple clustered damage, the relative frequencies of MBD (major groove base damage) and mBD (minor groove base damage) were higher, with more regular complexity distributions; (2) B‐DNA groove damage complexity is determined by the distribution density of groove damage points rather than their damage yield; (3) energy deposition in B‐DNA grooves predominantly depended on the groove damage complexity rather than the initial energy of the particle; (4) a mutual compensation mechanism existed between major and minor grooves, along with a difference in damage susceptibility. Findings demonstrate that although the distribution mechanism of groove damage in clustered B‐DNA damage is influenced by several factors such as proton linear energy transfer ( LET ), strand breaks in clustered damage, and DNA structure, it generally follows the four distribution characteristics described above. These findings provide a theoretical basis for optimizing proton‐targeted radiotherapy.