Double strand breaks (DSBs) of DNA molecules in tritiated water was examined under sterilized and non-sterilized conditions using a single molecule observation method. The genome DNA of bacteriophage T4 GT7 was immersed in sterilized tritiated water (5.2 MBq/cm3) and non-sterilized tritiated water (4.2 MBq/cm3) for 1, 7 and 14 day(s). Then the length of DNA molecules was measured using a fluorescence microscope after intercalation of fluorescent dye. The dose rate was 1.4–1.7 × 10−2 Gy/h and the dose level was 0.41–5.8 Gy. The rate of DSBs induced by β-rays from tritium was successfully evaluated under the sterilized conditions and the value comparable with the DSB rate under γ-ray irradiation (Noda et al., Scientific Reports 7 (2017) 8557) was obtained. The length of DNA molecules in non-sterilized tritiated water was clearly shorter than that in the sterilized tritiated water. This observation suggested that the effects of tritium was far weaker than that of microorganisms (e.g. bacteria) and impurities in water even at the tritium concentration as high as 5.2 MBq/cm3.