Sediment and freshwater mussel tissues were used to evaluate distribution and bioaccumulation of polynuclear (polycyclic) aromatic hydrocarbon (PAH) compounds in the lowermost Tennessee River and Kentucky Lake. The target analytes included naphthalene, phenanthrene, anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benz[a]pyrene, and benzo[g,h,i]perylene. Surface sediments (0–5 cm depth) from four locations in lowermost Tennessee River and five locations in Kentucky Lake were analyzed for target PAH compounds to determine spatial distributions. A sediment core from Ledbetter embayment in Kentucky Lake was analyzed to describe vertical distributions of the compounds. Freshwater mussels (Unionidae) collected from the lowermost Tennessee River and Kentucky Lake were analyzed and examined for bioaccumulation. PAH compounds were detected in all sediments and mussel tissues. PAH concentrations ranged from 0.01 (detection limit) to 90.4 ng/g dry wt in sediments and 0.02 (detection limit) to 223 ng/g dry wt in mussel tissue. Considering spatial distributions of PAHs in the sampling sites, there was no clear difference found between Kentucky Lake and the lowermost Tennessee River. Accumulation patterns of PAH species in sediment and mussel tissues exhibited the following order: Naphthalene > Phenanthrene > Benz[a]pyrene > Anthracene > Benzo[b]fluoranthene > Benzo[k]fluoranthrene > Benzo[g,h,i]perylene. The results revealed that 66.2% (w/w) were non-carcinogenic PAHs, whereas 33.8% (w/w) were carcinogenic in Kentucky Lake and the lowermost Tennessee River. In comparison with other freshwater ecosystems, PAH concentrations in Kentucky Lake and the lowermost Tennessee River were low.