Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.