During the dry season (May-October) in tropical northern Australia, the Flinders and Gilbert rivers dry into a series of in-stream pools, providing water for native fauna and stock and refugial habitat for terrestrial and aquatic biota. Despite streamflow in these rivers being strongly seasonal, with the vast majority of annual flow occurs during the wet season (November-April), their catchments have been identified as potential areas for further agricultural development. Thus water resource management plans should recognise any potential impacts that development and future climate may have on streamflow and dry-season pool persistence. We show how 8 years of Landsat imagery from 2003 to 2010 was used to derive a time-series of maps of in-stream waterbodies (67 to 97 dates per scene depending on cloud cover) and maps of pool persistence. Water-indices were derived for each Landsat scene and date and were calibrated to the extent of water mapped from high resolution imagery. The persistence of in-stream pools was estimated by calculating the percentage of time that a given pixel of set of pixels was mapped as water. Pools that existed for more than 90% of the time were identified as ‘key aquatic refugia’ and as such are likely to be crucial for sustaining ecosystems in these catchments. A much greater number of key aquatic refugia were found in the Gilbert catchment than in the Flinders catchment and this is anecdotally attributed to the greater role of groundwater in pool persistence in some parts of the Gilbert catchment. Such information, when related to stream flow rates and duration, are of importance in quantifying the impacts of changes in flow due to development of climate change.