In arid ecosystems, desert bighorn sheep are dependent on natural waterholes, particularly in summer when forage is scarce and environmental temperatures are high. To detect waterholes in Sierra Santa Isabel, which is the largest area of desert bighorn sheep habitat in the state of Baja California, Mexico, we used the normalized difference vegetation index (NDVI) and normalized difference water index (NDWI) from Sentinel-2 satellite images. Waterhole detection was based on the premise that sites with greater water availability, where NDVI was higher, can be identified by their density of vegetation greenness. For the detected waterholes, we estimated the escape terrain (presence of cliffs or steep, rocky slopes) around each by the vector ruggedness measure to determine their potential use by desert bighorn sheep based on the animals’ presence as documented by camera traps. We detected 14 waterholes with the NDVI of which 11 were known by land owners and 3 were unrecorded. Desert bighorn were not detected in waterholes with high values of escape terrain, i.e., flat areas. Waterhole detection by NDVI is a simple method, and with the assistance and knowledge of the inhabitants of the Sierra, it was possible to confirm the presence each waterhole in the field.