The Gray Fossil Site, northeastern Tennessee, is formed by multiple karst sub-basins filled with lacustrine sediments. The oldest sediments found were recently dated as Paleo-Eocene by palynological means, whereas the youngest sediments are considered Mio-Pliocene based on their faunal assemblage. In this study, we examined the Mio-Pliocene lacustrine sediments from the Gray Fossil Site to determine the Late Neogene floral characteristics of a site within the southern Appalachian Mountains. The Mio-Pliocene lacustrine sinkhole fill preserves a unique fossil assemblage, which includes invertebrate, vertebrate, and floral remains. Floral remains are represented by wood, seeds, leaves, and pollen grains. Forty-seven palynological samples from six different test-pits were analyzed. All pits exhibit a low pollen yield, a result of basic pH levels, drought, and fire events that occurred during deposition. The palynofloral assemblage has a low to moderate diversity and is largely dominated by a Quercus-Carya-Pinus assemblage (~ 90% of the palynoflora). The presence of Pterocarya grains supports a Late Neogene age for these lacustrine sediments. Comparison with modern pollen-based floras from North America suggests that: (1) examined pits can be discriminated into two separate groups based on their palynofloral signatures, (2) the Mio-Pliocene vegetation at the site ranged between a closed to open woodland setting, depending on the intensity and frequency of drought and fire events, and (3) the fossil palynofloral assemblage is comparable to what would be expected in the modern North American Mesophytic Forest region. © 2012 Elsevier B.V..