Nymphophilus minckleyi is a hydrobiid snail endemic to the freshwater spring ecosystem of Cuatro Ciénegas, Mexico. We used seven allozyme loci to examine the genetic substructure of N. minckleyi from 14 sites (subpopulations) in the basin and to test the hypothesis that spring pools in Cuatro Ciénegas are separated into seven hydrologically distinct drainages. Hierarchical F-statistics suggest significant population structure exists among the fourteen populations but not among the seven proposed drainages. Cluster analysis of Nei's genetic distance did not show populations grouping according to drainages, although it did reveal alternative clusters. We found two distinct morphotypes that were supported as genetically distinct groups by the allozyme data. Genetic studies of vagile species in desert spring ecosystems can be used to reveal hydrologic connections and identify genetically unique sub-populations.