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Waterholes are a key resource that influences wildlife distribution in semi-arid ecosystems. Mapping waterholes can guide intervening decisions for supplementing water resources and managing wildlife distribution patterns. Although remote sensing provides a key to mapping distribution of waterholes, efficiency of existing remotely sensed methods for detecting waterholes have to be evaluated and even new ones developed. In this study, we evaluated performance of the Modified Normalized Difference Water Index (MNDWI) and Superfine Water Index (SWI) at selected optimum thresholds. Kappa results indicated that MNDWI detects waterholes better than SWI. We further validated MNDWI detected waterholes by testing response of waterhole area to temporal rainfall variability and waterhole persistence to spatial rainfall variability. Extent of MNDWI-detected waterholes varied in relation to temporal rainfall variability (p<0.05). Waterhole persistence was not associated with spatial rainfall variability which could be explained by differences in waterhole types or low spatial rainfall variability.