North American paddlefishPolyodon spathulahistorically occurred in the Mississippi, Saint Lawrence, and Alabama rivers, as well as several other Gulf of Mexico coastal drainages. Recent population declines in some locations and the local extirpations of others purportedly have been due to habitat loss, overharvest, and other biotic and abiotic changes to riverine ecosystems. Mitigation for these declines has emphasized hatchery production and supplemental stocking programs, which use a number of geographical sources for parental stock. To investigate phylogeographic relationships within and among several watersheds, we surveyed allozyme and mtDNA variation among 189 individuals collected from six regions of the Mississippi River drainage plus the Mobile Bay and Pearl River drainages. Paddlefish exhibited lower levels of allelic and haplotypic diversity than other freshwater species, a characteristic shared with other “primitive” fishes. Allozyme variation at 64 presumptive loci was segregated into two major groups: a Mobile Bay drainage group and a group from the Mississippi River and Pearl River drainages. More subtle population divergences were observed among major regional tributaries within the Mississippi River, although a clear and unambiguous level of geographical clustering was not delineated. Furthermore, several of the localities harbored “private,” but rare, polymorphisms. These results demonstrate that the populations are sufficiently divergent to warrant differential management considerations for any rehabilitation, restoration, or protection measures.