Cyprinella caerulea (Blue Shiner) is a federally threatened minnow endemic to the Mobile Basin that is currently restricted to four disjunct populations. We examined the population structure in the Blue Shiner by sequencing the mitochondrial ND2 gene in 37 individuals. We recovered eleven haplotypes, with only one shared between populations, for an overall haplotype diversity of 0.768. Genetic differentiation between populations was significant, accounting for 26% of the variability found within the species. One individual morphologically identified as a Blue Shiner had a haplotype resolved with the sympatric Cyprinella trichroistia (Tricolor Shiner) in our phylogenetic analysis. Long-term management of the Blue Shiner should focus on restoring connectivity between populations in order to restore natural patterns of gene flow.