Freshwater mussels (Bivalvia:Unionidae) are among the most imperiled organisms in North America. Information on the spatial scale of reproduction and population connectivity will better enable mussel conservation programs to sustain long-term population viability, particularly restocking and recovery programs. Here we used genetic methods to characterize population structure, dispersal potential, and reproductive strategies in the freshwater mussel Lampsilis cardium from Twin Creek and Big Darby Creek (Ohio, USA). We genotyped adults and individual glochidia at 12 microsatellite loci to assess local population structure relative to within-population patterns of relatedness and parentage. Local populations within watersheds were weakly structured, and within-population estimates of relatedness identified probable full- and half-siblings several kilometers apart. Parent–offspring comparisons provided evidence of multiple paternity in single broods and identified the likely father of 3 glochidia from 1 female's brood 16.2 km upstream of the mother, indicating that long-distance transport of spermatozoa can promote population connectivity within watersheds. Given that lampsilines and other unionoids exhibit similar reproductive strategies, it is possible that other species are capable of long-distance fertilization. If so, fertilization in populations of many freshwater mussels might not be limited by local density of breeding adults. Therefore, the prospects for recovery of imperiled freshwater mussels might be better than what is now expected.