As oncological treatment might impair the patients' fertility, male cancer patients are offered to cryopreserve semen prior to treatment. Impaired sperm DNA quality is associated with reduced fertility, and in case of assisted reproduction, sperm DNA integrity may have an impact on choice of method. Therefore, we have assessed sperm DNA integrity in cancer patients, comparing pre- and post-treatment quality. Sperm DNA integrity was investigated in cryopreserved semen from 121 cancer patients, the predominating diagnoses were germ cell cancer (GCC) and Hodgkin's lymphoma (HL). Post-treatment samples, with a median follow-up of 3 years, were analysed for 58 of the men, allowing a pre- and post-treatment analysis on an individual basis. Sperm DNA integrity was assessed using the Sperm Chromatin Structure Assay and expressed here as the DNA Fragmentation Index (DFI%). One hundred and thirty-seven fertile men served as controls. Before treatment, GCC (n = 84) and HL (n = 18) patients had higher DFI% than controls (n = 143) with a mean difference of 7.7 (95% CI 3.2-8.8) and 7.0 (95% CI 2-12), respectively. The same trend was observed for other cancer diagnoses, but without reaching statistical significance (mean difference 3.6, 95% CI -1.2 to 8.4). No increase was seen in DFI% comparing pre- and post-treatment semen, regardless of treatment modality. A moderate elevation of DFI% was observed in cryopreserved semen from cancer patients. Oncological treatment, generally, did not induce any increase in DFI. These findings should be considered when discussing the utilization of pre-treatment cryopreserved semen vs. post-treatment fresh sperm in cancer patients undergoing assisted reproduction.