The goal of this study was to determine whether the quantification of radiation biomarkers in peripheral leukocytes of 111 breast cancer patients after adjuvant treatment with different modalities of three-dimensional conformal radiation therapy (3D-CRT) or intensity-modulated radiation therapy (IMRT) revealed any difference in the patients' radiation burden by out-of-field doses and an associated risk of second malignancies. Whole-breast radiation therapy was performed by 3D-CRT using either a hard wedge (n = 32) or a virtual wedge (n = 49) at dose rates of 3 and 6 Gy per min each. Patients receiving additional radiotherapy to lymph nodes were treated by 3D-CRT (n = 21) or IMRT (n = 9). DNA damage was measured as γ-H2AX foci (n = 111) and as unstable chromosomal aberrations (n = 15) in leukocytes drawn 30 min and 24 h after the first radiation fraction, respectively. The individual basal yield and radiation sensitivity ex vivo were assessed in leukocytes obtained before the first treatment. After radiation therapy, the average rate of γ-H2AX foci and chromosomal aberrations per leukocyte were dependent on multiple parameters of irradiation: the treatment volume, the administered equivalent whole-body dose, the number of monitor units and the beam-on time. Different modalities of radiation therapy caused significant variations in the levels of both radiation biomarkers irrespective of the treatment volume and administered dose, and in particular, a twofold higher rate after IMRT compared to 3D-CRT. Any deviation in biomarker response between radiation therapy techniques was directed by a linear dependence on the absolute beam-on time. However, the dispersion of γ-H2AX foci in peripheral leukocytes after radiation therapy correlated very well with the relative distribution of dose in the whole-body volume for each radiation therapy technique. In conclusion, the induction of radiation biomarkers in leukocytes of breast cancer patients by different radiotherapy modalities is dominated by general variables of irradiation. There was no significant difference in peripheral dose exposure observed in the investigated radiation therapy techniques. Radiotherapy techniques with prolonged absolute beam-on time increase the fraction of exposed leukocytes with pronounced risks for hematologic toxicities or immunosuppressive side effects.