White Sands pupfish (Cyprinodon tularosa) are endemic to southern New Mexico and occur in only four localities: Malpais Spring, Salt Creek, Mound Spring and Lost River. Recently reported historical accounts indicate that the latter two populations were derived from translocations. Their limited distribution and complicated history suggest that knowledge of population genetic structure would be useful for the development of a sound conservation strategy. Mitochondrial DNA (mtDNA) sequences for a segment of the control region showed little variation. Variation was observed for microsatellite and allozyme loci with 37% attributable to divergence among populations. The mean genetic distance between Malpais Spring and the other three populations was high (allozymes: Darc = 0.541; microsatellites: Rst = 0.684) compared with the mean distance among the other three populations (Darc= 0.161, Rst = −0.016). There were fixed or nearly fixed differences in allele frequency between the Malpais Spring population and the other three populations at one allozyme locus (hexokinase) and two microsatellite loci (WSP-02 and WSP-11). We suggest the recognition of two evolutionarily significant units (ESUs) for the White Sands pupfish: Malpais Spring and Salt Creek. Our data indicate that the Lost River and Mound Spring populations descended from translocations from the Salt Creek population. Therefore, conservation efforts should focus on the Malpais Spring population.