Amphibians draw great attention from biologists because of their importance as biomedical research models and conservation crisis, but housing and maintaining live animals is expensive and risky, especially as numerous transgenic and mutant lines are continually developed. The storage and management of cryopreserved germplasm can provide a safe and cost-effective alternative, reducing the number of live animals that need to be maintained to meet research demands. In collaboration with the Ambystoma Genetic Stock Center (AGSC), we are developing a reproducible cryopreservation pathway for the axolotl Ambystoma mexicanum. Although some studies have shown initial feasibility of cryopreserving A. mexicanum spermatophores, this method of collection is inconsistent, and standardized quality evaluation for in-vitro fertilization using thawed sperm has not yet been developed. We tested multiple factors and their interaction on sperm quality during storage and freezing, such as various osmotic pressures of an extender solution (Hanks’ balanced salt solution), different cryoprotectants (DMSO, DMFA, glycerol, methanol), and sugar additives (sucrose, trehalose). Based on this we have developed a practical pathway for in-vitro fertilization using thawed sperm collected through hormonal induction and abdominal massage. These results provide the groundwork for establishment and operation of an Ambystoma germplasm repository to protect, maintain, and distribute valuable genetic resources. This also has relevance to protection of genetic diversity of the small remaining wild populations of this species.