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Eighty-three paddlefish Polyodon spathula that were collected from 1995 to 1999 from the Missouri River Galvins Point Dam tailwater were analyzed for genetic variation in the mitochondrial DNA d-loop region. Additional samples from Montana, South Dakota, and Louisiana were used for comparative purposes. To facilitate the efficient analysis of numerous paddlefish samples, we applied a method that employs polyacrylamide gel electrophoresis (PAGE) to resolve restriction fragment length polymorphisms (RFLPs) amplified by polymerase chain reaction (PCR). DNA sequencing of 10 paddlefish revealed 22 polymorphic sites. Polymerase chain reaction–RFLP analysis of 93 paddlefish using three restriction enzymes detected six of the polymorphic sites and revealed six distinct haplotypes. All of the observed haplotypes were found in the Missouri River Galvins Point Dam tailwater. No temporal differentiation was observed among the 1995, 1998, and 1999 samples from the Missouri River Galvins Point Dam tailwater. Polymerase chain reaction–RFLP, resolved with PAGE, provided an efficient method for population genetic analysis of paddlefish.