Freshwater mussels (Bivalvia: Unionidae) are among the most imperiled organisms in North America. While there is an urgent need for effective conservation planning and management of these organisms, important basic biological information is lacking. This research characterizes levels of genetic diversity and population structure in the Near Threatened (IUCN) freshwater mussel Lampsilis cardium in populations from Twin Creek (southwest Ohio), Little Darby and Big Darby Creeks (central Ohio), and Little Muskingum River (eastern Ohio) and assesses the extent to which regional geological events explain population structuring. Data from the congener Lampsilis ovata from Clinch River (Tennessee) are included for comparison. To characterize these patterns, sequencing of the mtDNA gene cytochrome oxidase c subunit 1 (COI) and nuclear microsatellite genotyping over 12 loci was performed. Additionally, genotype data from both adults and individual glochidia were analyzed to examine parentage and within-population levels of relatedness across common pedigree relationships. Microsatellite data reveal weak population structuring across glaciated and unglaciated drainages and 900 total river miles. However, haplotype analysis and sequence alignment recovered deeply divergent, cryptic lineages within Lampsilis cardium consistent with ancestral introgressive hybridization with Lampsilis ovata or incomplete lineage sorting. Mito-nuclear discordance argues against ongoing hybridization, although polymorphic species are also consistent with the data, and this affirms the importance of multiple molecular markers. In addition to finding multiple paternity in single broods, a number of parent-offspring, full-sibling, and half-sibling relationships for adults and glochidia are described. Numerous instances are noted in which likely full-siblings or half-siblings were located several kilometers apart, demonstrating that DNA-based evidence can describe the spatial nature of dispersal in unionid mussels. In a first report, the likely father of three glochidia from one female's brood was identified 16.2 kilometers upstream, which suggests the possibility of long-distance transport of spermatozoa in Lampsilis cardium. Given the similarity with which Lampsilines reproduce, it is predicted that other members of this genus are also capable long-distance fertilization. If fertilization in populations of freshwater mussels is indeed not limited by the density of breeding adults, the prospects for recovery in this fauna may be better than recently imagined.