Nocomis leptocephalus (Bluehead Chub) is a minnow native to the southeastern United States that constructs nests used by many freshwater fishes. No microsatellite markers have been published for Bluehead Chub, and information on genetic structure and diversity is sparse. We evaluated microsatellites from other leuciscid species for use with Bluehead Chub and created a panel of markers that has sufficient power for investigations of population structure and can differentiate between Bluehead Chub and Notropis lutipinnis (Yellowfin Shiner) eggs. We applied the panel to Bluehead Chub samples from 2 locations in South Carolina, finding these populations are genetically differentiated with high levels of genetic diversity. Our marker panel can improve our understanding of population dynamics of Bluehead Chub and allow for informed conservation recommendations.