Given the continuing degradation of freshwater wetland ecosystems throughout the Southeast, there has been significant interest in developing methods and indices to evaluate and monitor wetland biological integrity. The purpose of this study was to adapt and test the ability of a vegetation-based assessment technique known as Floristic Quality Assessment to detect the level of human impact in hardwood flat wetlands of Southeastern Virginia. We measured plant species diversity and composition within each vertical stratum [herbaceous, woody understory (shrub and sapling), and canopy] of 11 wetlands. We calculated a Floristic Quality Index (FQI) for each layer, and tested for relationship to land use disturbance patterns within defined site buffer and watershed areas. We found floristic quality of the herbaceous layer and the sapling portion of the woody understory layer to be negatively correlated with level of land use disturbance at both buffer and watershed scales, suggesting that FQI scores within these strata reflect current anthropogenic stress. While FQI of the canopy layer and the shrub portion of the woody understory layer were not reliable indicators of current land use disturbance, we found that a comparison of sapling and canopy layer FQIs gave insights into historic vs. recent floristic integrity of sites. Overall, our findings support the use of floristic quality assessments in evaluating wetland biological integrity when sampling and index calculation methodology are carefully adapted to local flora and community types.