Floodplain wetland plant community resilience in semi-arid systems is attributed to their large, long-lived and persistent seed banks, and are a legacy of recent and historical water and land management regimes. Changes to these regimes influence seed bank dynamics resulting in seed gains or losses. In the Murray-Darling Basin river regulation has altered flooding regimes, requiring intervention with environmental flows which aim to maintain ecological structure and function of floodplain wetlands. But this requires an understanding of the patterns of seed bank composition at the landscape scale to inform their potential vulnerability to disturbance. As part of the Murray-Darling Basin Environmental Water Knowledge and Research (EWKR) project we investigated patterns of soil seed bank composition in relation to the flood frequency gradient (flooding nearly every year, every 1-3, 3-5 and 5-10 years) within structural vegetation types (non-woody wetlands, inland shrublands and inland woodlands) in the Macquarie Marshes, a large Ramsar listed semi-arid floodplain wetland. We found that the Macquarie Marshes have a large and diverse soil seed bank indicative of a high degree of resilience. Seed bank species richness was strongly influenced by flood frequency which is spatially heterogeneous across the Macquarie Marshes. Vegetation canopy modified the seed bank richness in locations of higher flood frequency. Anthropogenic disturbances may limit the resilience of the Macquarie Marshes by decreasing the flooding regime’s spatial heterogeneity and homogenising the seed bank. This has implications for water management decisions that prioritise inundation targets to maximise potential diversity. Our results also highlight the importance of environmental flows as they may create the only opportunity for seed bank replenishment during droughts, which are projected to become more frequent and protracted in the Murray-Darling Basin.