DNA samples are commonly frozen for storage. However, freezing can compromise the integrity of DNA molecules. Considering the wide applications of DNA molecules in nanotechnology, changes to DNA integrity at the molecular level may cause undesirable outcomes. However, the effects of freezing on DNA integrity have not been fully explored. To investigate the impact of freezing on DNA integrity, samples of frozen and non-frozen bacteriophage lambda DNA were studied using optical tweezers. Tension (5-35 pN) was applied to DNA molecules to mimic mechanical interactions between DNA and other biomolecules. The integrity of the DNA molecules was evaluated by measuring the time taken for single DNA molecules to break under tension. Mean lifetimes were determined by maximum likelihood estimates and variances were obtained through bootstrapping simulations. Under 5 pN of force, the mean lifetime of frozen samples is 44.3 min with 95% confidence interval (CI) between 36.7 min and 53.6 min while the mean lifetime of non-frozen samples is 133.2 min (95% CI: 97.8-190.1 min). Under 15 pN of force, the mean lifetimes are 10.8 min (95% CI: 7.6-12.6 min) and 78.5 min (95% CI: 58.1-108.9 min). The lifetimes of frozen DNA molecules are significantly reduced, implying that freezing compromises DNA integrity. Moreover, we found that the reduced DNA structural integrity cannot be restored using regular ligation process. These results indicate that freezing can alter the structural integrity of the DNA molecules.