Background The aim of the study was to evaluate the changes in γ-H2AX expression in peripheral blood lymphocytes (PBL) according to severity of radiation-induced mucositis. Patients and method Fifty patients with head and neck cancer treated with radiotherapy (RT) or chemoradiation were included in the study. Blood samples were collected before treatment to measure baseline γ-H2AX levels. Second sample was taken 45 minutes after the first RT fraction and then once a week, 45 min after irradiation. In patients treated with chemoradiation the blood sample was taken the day after chemotherapy. Mucositis was evaluated once a week and reported according to CTCAE v4 and RTOG/EORTC scales. PBL were analyzed with flow cytometry and level of H2AX phosphorylation at every time point was evaluated. Results In 35 patients mild to moderate (grade 1-2) mucositis was observed and 15 patients developed severe (grade 3) mucositis. No cases of grade 4 mucositis were observed. The difference in baseline levels of γ-H2AX between groups with mild and severe mucositis was statistically insignificant (p = 0.25). The statistically significant difference in γ-H2AX level was observed in week 7 of treatment (p = 0.01). No significant differences in γ-H2AX level were found neither between group treated with concomitant chemoradiation or RT alone neither between groups with and without common comorbidities. In the analysis of the kinetics of γ-H2AX during treatment, a statistically significant difference (p = 0.0088) between groups with mild and severe mucositis was observed. After fourth week of treatment levels of γ-H2AX decreased significantly in the group with severe mucositis and increased in patients with mild side effects. The observed difference was not caused by the decrease in peripheral lymphocyte count, which was similar in both groups. Conclusions Presented results indicate that severity of radiation-induced mucositis does not correlate directly with γ-H2AX levels measured in vivo in PBL. Prediction of mucositis grade based on γ-H2AX level is not yet possible, either before treatment or early during treatment, but preliminary results, indicating significant differences in γ-H2AX kinetics between groups, encourage further studies.