Background: Radiation therapy induces epigenetic changes that may influence treatment outcome. We investigated radiation-induced DNA methylation alterations as potential biomarkers in head and neck squamous cell carcinoma (HNSCC). Methods: Paired tumor samples from 45 HNSCC patients were collected before and during radiotherapy. Genome-wide methylation profiling was performed. Results: Distinct methylation changes occurred during treatment, with specific patterns associated with subsequent treatment response. Early methylation changes at specific loci correlated with local tumor control. Conclusions: Radiation-induced methylation dynamics may serve as early response biomarkers in HNSCC radiotherapy.