Nucleos(t)ide analogues (NAs) are currently the most important anti‑viral treatment option for patients with chronic hepatitis B (CHB). Adefovir dipivoxil (ADV), a diester pro‑drug of adefovir, has been widely used for the clinical therapy of hepatitis B virus infection. It has been previously reported that adefovir induced chromosomal aberrations (CAs) in the in vitro human peripheral blood lymphocyte assay, while the genotoxic mechanism remains elusive. To evaluate the possible mechanisms, the genotoxic effects of ADV on the TK6 and DT40 cell lines, as well as DNA repair‑deficient variants of DT40 cells, were assessed in the present study. A karyotype assay revealed ADV‑induced CAs, particularly chromosomal breaks, in wild‑type DT40 and TK6 cells. A γ‑H2AX foci formation assay confirmed the presence of DNA damage following treatment with ADV. Furthermore, Brca1‑/‑ DT40 cells exhibited an increased sensitivity to ADV, while the knockdown of various other DNA damage‑associated genes did not markedly affect the sensitivity. These comprehensive genetic studies identified the genotoxic capacity of ADV and suggested that Brca1 may be involved in the tolerance of ADV‑induced DNA damage. These results may contribute to the development of novel drugs against CHB with higher therapeutic efficacy and less genotoxicity.