The discovery and temporal persistence of xanthic phenotypes are reported for an isolated population of the twoline pupfish Cyprinodon bifasciatus, a species endemic to the Cuatro Ciénegas basin, México. During four observations over a 19 mo period, the Escobedita population of C. bifasciatus was found to harbor a low but stable frequency of xanthic individuals (recorded range 3.3–3.7%). Persistence of xanthic phenotypes in this system could reflect relaxed predation pressure, genetic drift in a small population, benign to favorable sexual selection or some combination of these factors. This population provides an exceptional natural laboratory for investigation of evolutionary influences on the frequency of xanthic variants in wild populations.