Background: Approximately 20% of oral squamous cell carcinoma (OSCC) cases arise without any identifiable environmental cause, suggesting involvement of genetic influences in their aetiology. DNA double-strand breaks (DSBs) sever both strands of DNA and pose a potential threat to genomic integrity. A hastened accumulation of somatic mutations consequent to DSB repair is deemed to be a likely event in tumorigenesis of OSCC. Methods: Two discrete chemical approaches, namely hydrogen peroxide and camptothecin, were used to induce DSB in oral cell lines derived from normal through dysplastic to OSCC tissues. After optimization, gamma histone 2Ax (γH2Ax) foci were counted as an indirect measure of kinetics of DSB and confirmed with Western blot of γH2Ax, Nbs1 and ATM. Results: Maximal number of γH2Ax foci was detected 1 and 2 hours post-exposure to camptothecin and hydrogen peroxide, respectively; when adjusted for the baseline number of γH2Ax, neoplastic cell lines showed the lowest number of maximal DSB and slowest rate of repair compared to other cell lines. γH2Ax Western blot closely mirrored the trend observed in immunofluorescent staining for γH2Ax foci. Changes in the expression level of ATM and Nbs1 were minimal; however, ATM expression showed a slight gradual increase in normal cells which reached its peak at 2 hours after exposure to camptothecin. Conclusions: There is a difference in efficiency of DSB repair pathways in cell lines derived from different stages of oral tumorigenesis with neoplastic cell lines having the most defective DSB repair system.