Robust Redhorse (Moxostoma robustum) is a deep-dwelling catostomid that lives in southeastern rivers of the United States. After being lost to science for about 100 years, M. robustum was rediscovered in the 1980s by the Georgia DNR and is a federal species of special concern. Our goal was to genetically characterize populations of M. robustum in the Savannah and Pee Dee rivers based on genetic diversity and population structure using a suite of ten microsatellites. Substantial levels of population structure were detected between rivers (R_ST  =  0.308), with high genetic diversity (0.81–0.87) and low inbreeding in both systems. Long-term estimates of effective population sizes were very high for both systems, but contemporary estimates were substantially lower, with the Pee Dee River estimate being of concern from a conservation standpoint. Their long life span and overlapping generations result in a high potential for across-year-class spawning and are likely contributing to the maintenance of high genetic diversity in light of their decreased effective population sizes. Although statistical modeling indicates a faster loss of allelic richness as compared to heterozygosity, results are congruent as the Savannah River population is forecast to maintain 90% of its genetic diversity for >200 years and the Pee Dee for ∼65–120 years. These results support the current management view of each river as evolutionarily significant units, provide an important genetic baseline for future monitoring of populations of M. robustum, and build an excellent foundational tool for any future population restoration activities.