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Refuge populations of Cyprinodon macularius and Cyprinodon eremus, the extant members of the endangered desert pupfish complex, have been maintained for up to 33 years in semi-natural refuges. We examined the success of the refuge program in maintaining diversity at four microsatellite DNA loci in 24 refuge populations of C. macularius and six of C. eremus that include, respectively, seven and four lineages representing original translocations from the wild. These lineages have been maintained with essentially no inoculations of genetic material from the wild and, except for one refuge, no intermixing of lineages. Comparison with wild-source populations showed marked declines in diversity within local refuges and within lineages, but relatively minor declines for the composite of all refuge populations for each species. In genetic makeup, the refuge populations generally clustered by lineage, indicating significant genetic drift early in lineage history. The results indicate that, with relatively minor adjustments in management, the refuge program can successfully preserve a large portion of the wild genetic diversity in the desert pupfish complex.