The Bayou Darter, Etheostoma rubrum, is endemic to the Bayou Pierre system of southwestern Mississippi where it occupies swift, shallow riffles with coarse, firm substrata. The Bayou Pierre system has experienced extensive erosion in response to rapid headcutting leading to loss of riffle habitat and degradation of riverine conditions. Due to its high degree of habitat specificity along with ongoing habitat fragmentation and potentially reduced gene flow between isolated populations, the Bayou Darter is vulnerable to severe population declines and possible extinction. Our objectives were to quantify levels of genetic diversity in the mitochondrial control region and infer population structure across the range of the species. Sequencing of 106 sampled individuals revealed only three mtDNA haplotypes with one variable site. Haplotype diversity and nucleotide diversity were low (h ≤ 0.11 and π < 0.001), and there was no structure revealed among the four sampled populations. The low genetic diversity in E. rubrum may best be explained by a small range size and recent genetic bottleneck.