Long-lasting surface water in arid-lands provide oases for aquatic biota, but their values as biological refugia have rarely been assessed. This study identified andmappedpermanent natural wetlands across the Eastern Lake Eyre Basin in Australia and classified them into four types: riverine waterholes, rockholes, discharge springs and outcrop springs. Waterholes are the most widespread and numerous source of lasting water, while springs and rockholes are confined to relatively discrete clusters. The characteristics of each wetland type are summarised, and their biological values compared by examining various scales of endemism for vascular plant, fish and mollusc species. Discharge springs contain an exceptional concentration of endemic species across all three lifeforms at a range of scales. Waterholes are critical drought refugia for native fish species that also utilise a vast network of ephemeral streams during and after floods. Rockholes and outcrop springs do not contain any known specialised endemics, although the latter have disjunct populations of some plants and fish. The existing knowledge of antiquity, connectivity and habitat differentiation of the wetland types is compiled and their role in determining biological endemism is discussed. Exotic fish are a major conservation issue, the recovery of the discharge springs should be paramount, and the intact network of permanent waterholes should be preserved. A focus on endemism, combined with an understanding of the biogeographical processes underlying the observed patterns provides an effective and systematic approach to setting priorities for regional biodiversity conservation.