ABSTRACT: Habitat fragmentation reduces gene flow and population resiliency. Fragmentation can contribute to localized extinctions, and for small-range taxa, range-wide extinction. Accelerated erosion in streams and rivers may fragment habitats for habitat-specialist species by greatly altering river mainstems, pushing many taxa into isolated tributaries. In this paper we examine patterns of genetic structuring in the federally threatened and endemic Bayou Darter Nothonotus rubrus to assess whether patterns of erosion are causing fragmentation of populations. Patterns of heterozygosity support the hypothesis of recent colonization of upper White Oak Creek. STRUCTURE analysis yielded unexpected results: a population isolated above an erosional feature, a population widespread throughout the watershed, and evidence of introgression between 2 of 3 genetic clusters. Multivariate analyses support STRUCTURE in suggesting evidence for three genetically distinct populations. Effective population size analysis found estimates of abundance to be spatially concordant with recent field studies. Our results demonstrate unexpected population structuring in N. rubrus, potentially due to populations becoming separated by features related to in-channel erosion. The amount of local adaptation related to this differentiation is unknown; however, conservation efforts and efforts to augment existing populations should take into account genetic variation and potential local adaptation. We recommend next steps for further exploring genetic variation and potential conservation strategies for this species.