Mitochondrial and nuclear gene variation is described from populations representing the range of two species of rare pygmy sunfishes, Elassoma boehlkei and Elassoma okatie, in the Carolinas. Mitochondrial control region sequences provide evidence for a minimum of four Evolutionarily Significant Units (ESU), two each within E. boehlkei and E. okatie. Significant allele frequency differences at a single nuclear gene locus corroborate the distinctiveness of these ESUs. Phylogenies based on control region haplotypes and frequencies of nuclear alleles are inconsistent with the currently described range of E. boehlkei and E. okatie. Despite inconsistency with described species boundaries, the result of this genetic analysis for rare species management is clear: conservation strategies that describe distinct population segments inhabiting individual river systems should be encouraged.