Persistent seed banks are a key plant regeneration strategy, buffering environmental variation to allow population and species persistence. Understanding seed bank functioning within herb layer dynamics is therefore important. However, rather than assessing emergence from the seed bank in herb layer gaps, most studies evaluate the seed bank functioning via a greenhouse census. We hypothesise that greenhouse data may not reflect seed bank-driven emergence in disturbance gaps due to methodological differences. Failure in detecting (specialist) species may then introduce methodological bias into the ecological interpretation of seed bank functions using greenhouse data. The persistent seed bank was surveyed in 40 semi-natural grassland plots across a fragmented landscape, quantifying seedling emergence in both the greenhouse and in disturbance gaps. Given the suspected interpretational bias, we tested whether each census uncovers similar seed bank responses to fragmentation. Seed bank characteristics were similar between censuses. Census type affected seed bank composition, with >25% of species retrieved better by either census type, dependent on functional traits including seed longevity, production and size. Habitat specialists emerged more in disturbance gaps than in the greenhouse, while the opposite was true for ruderal species. Both censuses uncovered fragmentation-induced seed bank patterns. Low surface area sampling, larger depth of sampling and germination conditions cause underrepresentation of the habitat-specialised part of the persistent seed bank flora during greenhouse censuses. Methodological bias introduced in the recorded seed bank data may consequently have significant implications for the ecological interpretation of seed bank community functions based on greenhouse data.