Background: Radiotherapy is regarded as a standard treatment modality in breast cancer (BC). Radiation causes cellular damage both in cancer and normal cells by inducing DNA-damage and chromosomal aberrations (CA). Different agents were used for ameliorating effects of radiation, mainly antioxidants such as melatonin. Melatonin shows oncostatic properties on human BC. The aim of this study was to evaluate the modulating effect of melatonin on radiation induced CA in cells irradiated at G2 phase of the cell cycle. Materials and methods: G2 assay was applied on whole peripheral blood lymphocytes received from 10 BC patients and 5 normal controls. Blood culture was initiated in complete culture medium. Four h prior to harvesting, cells were irradiated with 1Gy gamma rays. Pretreatment of samples with melatonin was done 3 h before irradiation. After metaphase preparation and slide making, slides were stained in Giemsa. Hundred well spread metaphases were scored for the presence of chromatid type aberrations with a microscope at a magnification of × 1000. Result: Results indicated a high and significant frequency of CA both in lymphocytes of normal and breast cancer patient after irradiation; however, the frequency was much more in lymphocytes from BC patients. Pretreatment of samples with melatonin led to a considerable increase in the frequency of aberrations especially in lymphocytes of BC patient. Conclusion: Results showed that despite having antioxidant property, melatonin led to enhanced frequency of radiation induced CA in lymphocytes of BC patients.