Freshwater mussels (Bivalvia: Unionidae) are the most imperiled groups of organisms in North America. Accurately defining species boundaries is vital for ongoing biological conservation and management efforts. The false spike or Fusconaia mitchelli (Simpson in Dall, 1896) is a freshwater mussel found in the Brazos, Colorado, and Guadalupe drainages. A previous molecular study depicted significant intraspecific genetic variation which may be indicative of speciation occurring between geographically separated populations. Here, we use multi-locus sequence data to re-evaluate systematic relationships between F. mitchelli in the three central Texas drainages. We sequenced both mitochondrial (mtDNA) and nuclear loci: the protein-coding mtDNA genes cytochrome oxidase subunit 1 and NADH dehydrogenase 1, and the nuclear ribosomal internal transcribed spacer 1. Phylogenetic and phylogeographic analyses depict molecular divergence between two groups within F. mitchelli coinciding to the Guadalupe drainage, and the Brazos and Colorado drainages. The junior synonym Fusconaia iheringi is available for the Brazos and Colorado populations and elevating it from synonymy warrants further investigation using an integrative taxonomic approach.