Cryopreservation of genetic material can become an important tool for user groups in imperiled fishes, wild fisheries, aquaculture, and biomedical research. Persistent challenges within aquatic species cryopreservation are standardization and reliable collection of diverse, high quality samples. The overall goal of this study was to work with different user groups and cryopreserve sperm on-site at their facilities to evaluate the uses and challenges of a mobile laboratory with high-throughput and quality control capabilities comparable to those of a specialized centralized facility. The objectives were to demonstrate collection and cryopreservation of sperm of: 1) large-bodied freshwater Blue Catfish (Ictalurus furcatus) for aquaculture; 2) small-bodied freshwater Xiphophorus for biomedical and imperiled repository development, and 3) saltwater Red Snapper (Lutjanus campechanus) for wild fisheries research. Over the course of this project, the mobile laboratory traveled more than 4,000 km collecting germplasm from more than 650 male fishes. A total of 136 Blue Catfish were processed in 2015 and 2016 resulting in a total of 6,146 0.5-mL French straws. A total of 521 males from 11 different species in the genus Xiphophorus were processed over 4 d in 2015 resulting in a total of 488 0.25-mL French straws. And, a total of 17 Red Snapper males were processed during 2015 resulting in a total of 316 0.5-mL French straws. This is the first development of a mobile laboratory with high-throughput capability for aquatic species. User groups would no longer be limited to germplasm resources that can only be shipped as samples or transported as live animals to a central cryopreservation facility. Mobile laboratories create opportunities to collect higher quality germplasm, provide access to new species, and enable direct cooperation, including training, with a wide variety of user groups and applications.