Wetlands are biodiversity hotspots, providing refuge to threatened species globally, and diverse ecosystem services for humans. Wetland values are threatened by changes to hydrological regimes caused by anthropogenic degradation and climate change. Utilizing improved, large-scale datasets to characterize regional trends in wetland stress allows us to identify regions of concern and prioritise conservation and adaptation responses. We developed a novel approach to characterizing wetland hydrologic stress that accounts for natural variability in wetland water regimes. Our approach integrated remotely sensed inundation observations over the period 1988 to 2018 to enable a large-scale assessment of ∼ 37,000 wetlands in south-east Australia. We assessed stress in six hydrologic inundation regime metrics: mean magnitude, maximum magnitude, duration, timing, frequency, and rate of change. In wetlands across the region, we observed a general decreasing trend over the historical period 1988 to 2018 in four of six stress indices: magnitude (mean), magnitude (max), duration, and frequency. More than 20 % of wetlands had a stress index of ± 1 for magnitude (mean) and duration, indicating significant departure from the existing hydrologic regime. These results are consistent with the impacts of observed reductions in cool season rainfall, which supports evidence that climate change has already impacted this region. The findings reinforce the importance of wetland management under a future changing climate. Our approach can be used to characterize trends in wetland hydrologic stress globally, given the compatibility with remotely sensed data. Management agencies will benefit from using these approaches to better understand how wetland hydrology is shifting across regional scales, and where to prioritise their resources to persist, adapt or transform wetlands under continued future change.