We evaluated methodology for estimating total plant species richness in seasonally inundated depressional wetlands of the longleaf pine (Pinus palustris Mill.) ecosystem. Our objectives were to assess non-parametric estimators of plant species richness based on species incidence plot samples and to determine the minimum sampling requirement for accurate extrapolation of species richness. We quantified species accumulation in varying sample plot areas and spatial distribution within nine depressional wetlands. We recorded species presence on 1 m², 4 m², and 100 m² plots along transects extending across each wetland. Increasing sample area from 1 m² to 4 m² plots resulted in an increase in mean species richness up to 33%. EstimateS was used to estimate total species richness for each wetland and sampling method. Species accumulation curves did not reach a plateau and none of the estimates showed a convergence of estimated and observed richness. Due to the large number of unique species and the heterogeneous distribution of species in these hyperdiverse wetlands, we conclude that the most commonly used non-parametric estimators are not effective as tools to obtain accurate estimates of total species richness.