The freshwater mussel genus Obovaria (Bivalvia: Unionidae) has been recognized as taxonomically challenging due to difficulties distinguishing intraspecific variation in shell shape from interspecific convergence. Here we provide details of molecular data used in an integrative approach to investigate species boundaries in Obovaria in the Gulf Coastal Plain and adjacent drainages. The details include DNA sequences from two mitochondrial genes (COI, ND1) and one nuclear locus (ITS1), along with collection details, museum catalog numbers, and GenBank accession numbers. We provide the molecular data in multiple formats to facilitate future research involving freshwater mussel taxonomy, evolution, identification, and environmental DNA projects.