We used starch gel electrophoresis to assay 24–28 presumptive gene loci in captive and natural populations of three species of endangered fishes held at the Dexter (New Mexico) National Fish Hatchery. The species included two cyprinodontids, the Leon Springs pupfishCyprinodon bovinusand Comanche Springs pupfishC. elegans, and a poeciliid, the Pecos gambusiaGambusia nobilis. Hatchery stocks had been founded with 30–80 wild fish and subsequently held in captivity for 6–8 years in 0.1–0.2-hectare ponds where spawning has occurred spontaneously. Heterozygosity and polymorphism of the hatchery stocks were similar to those of the populations from which they were derived. However, the captive populations apparently had lost some rare alleles, probably due to the small sizes of the founding populations. Managers should avoid complacency with small species that spawn spontaneously in hatcheries and, superficially, seem to require little special attention. Minimum requirements for management of such species include initiating captive stocks with hundreds of individuals from natural populations, monitoring genetic status and population size of captive stocks, and, where possible, periodically inoculating genetic material from natural populations.