Although thousands of genetically modified mouse strains have been cryopreserved by sperm freezing, the likelihood of cryorecovery success cannot be accurately predicted using conventional sperm parameters. The objective of the present study was to assess the extent to which measurement of a sperm DNA fragmentation index (DFI) can predict sperm quality and fertility after cryopreservation. Using a modified TUNEL assay, we measured and correlated the DFI of frozen-thawed sperm from 83 unique mutant mouse strains with sperm count, motility and morphology. We observed a linear inverse correlation between sperm DFI and sperm morphology and motility. Further, sperm DFI was significantly higher from males with low sperm counts compared to males with normal sperm counts (P < 0.0001). Additionally, we found that viable embryos derived using sperm from males with high DFI (62.7 ± 7.2% for IVF and 73.3 ± 8.1% for ICSI) failed to litter after embryo transfer compared to embryos from males with low DFI (20.4 ± 7.9% for IVF and 28.1 ± 10.7 for ICSI). This study reveals that measurement of DFI provides a simple, informative and reliable measure of sperm quality and can accurately predict male mouse fertility.