The purpose of this study was to evaluate DNA damage and chromosomal aberrations in peripheral blood lymphocytes (PBLs) by chemoradiotherapy (CRT) in patients with esophageal cancer and to figure out the relationship between DNA damage and toxicities by CRT. Twelve esophageal cancer patients who received CRT were enrolled in this study. Prescribed doses were 60 Gy in 9 patients and 50 Gy in 3 patients. Patients received 2 Gy radiotherapy (RT) every 24 hours five days a week. Cisplatin (days 1 and 29) and 5-fluorouracil (days 1–4 and 29–32) were used as chemotherapy. We obtained PBLs just before and at 15 minute after RT on the day when the cumulative dose reached 2 Gy, 20 Gy, 40 Gy, and 50 or 60 Gy (the last day of RT). PBLs were also obtained at 4 weeks and 6 months after RT from all and five patients, respectively. Dicentric and ring chromosomes in PBLs were counted using fluorescence in situ hybridization analysis method with centromere and telomere peptide nucleic acid probes (PNA-FISH) to evaluate the number of chromosomal aberrations for each cumulative dose. γH2AX foci per cell were counted and the increment of γH2AX foci by 2 Gy RT for each day was calculated to assess DNA double-strand breaks. Adverse events were evaluated using common terminology criteria for adverse events version 4.0. Students t test was used to compare the mean value between two groups and p value < 0.05 was considered statistically significant. The mean numbers of chromosomal aberrations per 1,000 cells were increased during CRT as follows; Before RT: 18 (range: 0 – 53), 2 Gy: 80 (49 – 268), 20 Gy: 529 (288 – 1010), 40 Gy: 749 (392 – 1333), the last day of RT (50 Gy or 60 Gy): 1217 (794 – 2296), 4weeks after RT: 1128 (455 – 2063) and 6 months after RT: 512 (388 – 581). On the other hand, the mean number of γH2AX foci per cell before RT showed no increase during CRT. The mean increment of γH2AX foci per cell by 2 Gy RT was significantly reduced as follows; 2 Gy: 2.57 (1.01 –3.75), 20 Gy: 2.48(1.18 –3.85), 40 Gy: 1.52 (0.07 – 2.84), the last day of RT (50 Gy or 60 Gy): 1.34 (0.44 – 3.47), even though the same dose was administered each day. Four out of twelve patients showed grade 3 toxicities (three esophagitis and one pneumonitis) during or after CRT. The numbers of chromosomal aberrations on the last day of RT were significantly higher in those with grade 3 toxicities than those without grade 3 or worse toxicities. (mean value: 1609 vs 851, p=0.007) The mean increment of γH2AX foci by 2 Gy RT on the last day was also significantly higher in those with grade 3 toxicities than those without grade 3 or worse toxicities. (mean value: 2.20 vs 0.75, p=0.008) The number of chromosomal aberrations after CRT increased as the cumulative doses increased, while the increment of γH2AX foci decreased with an increase in cumulative dose. The numbers of chromosomal aberrations and the increment of γH2AX foci on the last day of RT seemed to correlate the severity of toxicities by CRT for esophageal cancer patients.