Allelic variation at hypervariable, nuclear-encoded loci and mitochondrial (mt)DNA was studied among three geographic samples (40 individuals) of the critically endangered Cape Fear shiner, Notropis mekistocholas. Genetic variation, as measured by allelic richness and gene (microsatellite) or nucleon (mtDNA) diversity, was similar to that in other fish species. Homogeneity tests of allele and genotype distributions and analysis of molecular variance (amova) at nuclear-encoded loci revealed significant genetic heterogeneity among localities. No differences in mtDNA allele (haplotype) frequencies were detected. The ratio of the number of microsatellite alleles to the range in allele size suggested that significant reductions in effective size have occurred at two of the three localities. Long-term (inbreeding) effective population size differed among the samples and ranged from ∼1,300 to ∼3,000. Collectively, these results indicate that (i) Cape Fear shiners at these localities are not genetically impoverished, (ii) separate populations of Cape Fear shiners may exist in the Cape Fear drainage, (iii) recent reduction in effective size may have occurred in two of the three localities, and (iv) ancestral populations of Cape Fear shiners may have been of sufficient effective size to offset extinction due to genetic factors.