Spermatogonia cryopreservation is a method to preserve valuable genomes from both maternal and paternal origin. The damage associated with the application of this technology on post-thaw cell quality is important to assess, including at the epigenetic level. This study aimed to assess post-thawed spermatogonia quality by evaluating alterations in plasma membrane integrity, DNA integrity (fragmentation and apoptosis), lipid peroxidation (malondialdehyde levels) and epigenetic modifications (DNA methylation profile). We observed that plasma membrane integrity (fresh 78.98 % ± 5.66; cryopreserved 62.81 % ± 3.25; P = 0.003) and DNA integrity (fresh 32.95 % ± 2.28; cryopreserved 37.28 % ± 1.87; P = 0.0026) were affected by cryopreservation, while no difference in lipid peroxidation was observed (fresh 1.13 % ± 0.45; cryopreserved 0.91 % ± 0.96; P = 0.701). While global levels of DNA methylation were unaffected by cryopreservation (fresh 82.80 % ± 0.47; cryopreserved 83.32 % ± 0.81; P = 0.745), some differentially methylated cytosines (DMC) were observed in cryopreserved versus fresh spermatogonia (156 DMC). This study showed that spermatogonia cryopreserved according to our protocol provides a good supply of undamaged cells for several applications. The significance of the few detected DMCs deserves further attention since it may affect gamete differentiation and epigenetic profile.