The restoration and recovery of imperiled mussel species will require the re-establishment of populations into historically occupied habitats. The possible existence of genetic differentiation among populations should be considered before inter-basin transfers are made. Eighty individuals of the federal candidate species Lexingtonia dolabelloides were sampled from populations in the North Fork Holston, Middle Fork Holston, Clinch, Paint Rock and Duck rivers of the Tennessee River basin in the southeastern United States. We sequenced 603 base-pairs of a mitochondrial DNA gene (ND-1) and 512 base-pairs of a nuclear DNA gene (ITS-1). Analyses of molecular variation (AMOVA) values for both genes indicated that the majority of variation in L. dolabelloides resided within populations (82.9-88.3%), with 11.7-17.1% of variation among populations. Haplotype frequencies differed significantly among populations for both genes sequenced. Clustering of haplotypes in minimum-spanning networks did not conform stringently to population boundaries, reflecting high within-population and low between-population variability. Maximum parsimony analysis did not identify any population as a monophyletic lineage. A Mantel test showed no significant correlation between geographical stream distance and genetic distance, thus not supporting a pattern of isolation-by-distance. Overall, results provided support to manage fragmented populations of L. dolabelloides in the Tennessee River drainage as two management units (MUs), but did not provide evidence for the existence of ESUs following published molecular criteria. ?? The Author 2005. Published by Oxford University Studies on behalf of The Malacological Society of London, all rights reserved.