This study aims to explore the temporal pattern of DNA breaks induced by nanosecond electric pulses (nsEP) in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Human ovarian cancer cells A2780 (cisplatin-sensitive subline) and C30 (cisplatin-resistant subline) were exposed to nsEP. Sham exposed groups were shame exposed to nsEP. Cell viability was determined using CCK-8 assay after 0 h, 4 h, 8 h, 12 h and 24 h, respectively, and the percentage of dead cells was calculated. The DNA break was detected with the alkaline single cell gel electrophoresis (comet assay), and the 75th percentiles of TL (tail length), TM (tail moment) and OTM (Olive tail moment) were measured. Cell viability displayed an early decrease and late increase, with the valley value seen at 8 h. Percentages of cell death and comet-formed in A2780 cells were higher than those in C30 cells (P<0.05) at 8 h, respectively. TL, TM and OTM in C30 cells were less than those in A2780 cells (P<0.05). The percentage of comet-formed correlated with that of cell death in either A2780 (r=0.997, P<0.05) or C30 (r=0.998, P<0.05) cells. DNA breaks induced by nsEP in cisplatin-sensitive cells differred from that in resistant cells, and DNA break resulted in fraction of cell death.