A Memorandum of Understanding, which formed the Robust Redhorse Conservation Committee (RRCC), was developed and adopted by concerned stakeholders in Georgia, South Carolina, and North Carolina to attempt recovery of the recently “rediscovered,” and rare, robust redhorse Moxostoma robustum. As part of the conservation program, genetic analyses of robust redhorse populations were needed to facilitate responsible hatchery programs that could be used to supplement natural reproduction in declining populations and to reestablish populations in rivers in which it may have historically been present. We analyzed mtDNA control region sequences in robust redhorse from the Oconee and Ocmulgee Rivers in the Altamaha River drainage and the Savannah River to determine the genetic relatedness of these populations to guide restoration efforts. Fixed differences in mtDNA haplotypes were found between robust redhorse from the two drainages that strongly argues for the designation of these populations as Evolutionarily Significant Units and against the interstock transfer of fish. Further studies will be needed to evaluate the full significance of these differences and associated management implications.