Freshwater mussels (Unionidae) are among North America's most imperilled organisms. Mussels produce small larvae (glochidia) that parasitize aquatic vertebrates. We modified the Epicentre QuickExtract protocol to extract DNA from a single glochidium, collected directly from the marsupium of a female mussel, to use as template in polymerase chain reactions (PCRs). Yield per glochidium in a 40 µL extraction volume provided enough DNA for ≥ 15 PCRs per individual. We were successful in using this DNA for microsatellite analysis of up to three loci per individual. Offspring from one female showed evidence for multiple paternity within her brood. Our results are the first documentation of this phenomenon in freshwater mussels.