The Iowa Pleistocene snail, Discus macclintocki, is an endangered species that survives only in relictual populations on algific (cold-air) talus slopes in northeast Iowa and northwest Illinois in the central region of the USA. These populations are believed to have been isolated since the temperatures began to warm at the end of the last glacial period around 16 500 years ago. DNA sequencing of the 16s rRNA gene of the mitochondria was used to determine the genetic relationship among 10 populations and the genetic diversity within these populations. Genetic diversity is extremely high within this species with 40 haplotypes spread across the 10 populations sampled within a 4000 km2 region. Phylogenetic analyses showed that haplotypes formed monophyletic groups by the watershed on which they were found, suggesting that watersheds were important historical avenues of gene flow. Genetic distances were strongly related to the geographical distance among all populations, but this relationship was dependent on the scale being considered.