In this study, a non-lethal DNA isolation technique was developed for freshwater mussels. A total of 45 Quadrula quadrula, 21 Q. metanevra, and 19 Q. pustulosa were collected from the Neosho and Verdigris Rivers in eastern Kansas. DNA was successfully isolated from 82 mussels using the non-lethal technique developed in this study. Spectrophotometer analysis of the 82 DNA samples resulted in an average DNA concentration of 488 ng/μl. Agarose gel electrophoresis of polymerase chain reaction products obtained using the purified mussel DNA demonstrated high quality, reproducible amplification products. All mussels remained alive for several months after tissue biopsy in the laboratory. A mark and recapture study was also performed to confirm survivability, thereby ensuring our procedure was non-lethal. Recapture efforts resulted in a 56% live recovery of mussels from the Neosho River and a 78% live recovery from mussels in the Verdigris River approximately four and one-half months after their release.