Estimating species richness with herbarium data and new collections allows us to understand the distribution of diversity. We investigated the accuracy of lycophyte and fern sampling along a vegetation gradient in the subtropical Atlantic Forest in southern Brazil. We compiled lycophyte and fern collection metadata and estimated species richness and assessed sampling accuracy for sixty 50 x 50 km units using ACE, Chao 1, Chao 2, Jackknife 1 and Jackknife 2 estimators. We compiled data for 12,779 fern specimens of 441 species, 67 of which were sampled in only one unit (singletons) and 35 in two units (duplicates). Of the 60 units examined, only 11 had observed values that were above 70% of their estimated values, and 14 had observed levels between 65-70% of the estimated values, meaning that 35 units had a sampling accuracy of less than 65%. In spite of the long history of lycophyte and fern collecting in the study area, there remain units with a lower than expected sampling accuracy for a subtropical forest. is nding indicates that a sizeable collection e ort is needed in order to discover the actual distribution of species before the e ects of fragmentation and deforestation become permanent.