Molecular data have been an integral tool in the resolution of the evolutionary relationships and systematics of freshwater mussels, despite the limited number of nuclear markers available for Sanger sequencing. To facilitate future studies, we evaluated the phylogenetic informativeness of loci from the recently published anchored hybrid enrichment (AHE) probe set Unioverse and developed novel Sanger primer sets to amplify two protein-coding nuclear loci with high net phylogenetic informativeness scores: fem-1 homolog C (FEM1) and UbiA prenyltransferase domain-containing protein 1 (UbiA). We report the methods used for marker development, along with the primer sequences and optimized PCR and thermal cycling conditions. To demonstrate the utility of these markers, we provide haplotype networks, DNA alignments, and summary statistics regarding the sequence variation for the two protein-coding nuclear loci (FEM1 and UbiA). Additionally, we compare the DNA sequence variation of FEM1 and UbiA to three loci commonly used in freshwater mussel genetic studies: the mitochondrial genes cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1), and the nuclear internal transcribed spacer 1 (ITS1). All five loci distinguish among the three focal species (Potamilus fragilis, Potamilus inflatus, and Potamilus purpuratus), and the sequence variation was highest for ND1, followed by CO1, ITS1, UbiA, and FEM1, respectively. The newly developed Sanger PCR primers and methodologies for extracting additional loci from AHE probe sets have great potential to facilitate molecular investigations targeting supraspecific relationships in freshwater mussels, but may be of limited utility at shallow taxonomic scales.