Abstract: The environmental risks from shale gas extraction through the unconventional method of ‘fracking’ are considerable and impact on water supplies below and above ground. Since 2010 the recovery of natural shale gas through fracking has been proposed in parts of the fragile semi-arid ecosystems that make up the Karoo biome in South Africa. These unique ecosystems are heavily reliant on underground water, intermittent and ephemeral springs, which are at great risk of contamination by fracking processes. Diatoms are present in all water bodies and reflect aspects of the environment in which they are located. As the possibility of fracking has not been removed, the aim of the project was to determine if diatoms could be used for rapid biomonitoring of underground and surface waters in the Karoo. Over a period of 24 months, water samples and diatom species were collected simultaneously from 65 sites. A total of 388 diatom taxa were identified from 290 samples with seasonal and substrate variation affecting species composition but not the environmental information. Species diversity information, on the other hand, often varied significantly between substrates within a single sample. Analysis using CCA established that the diatom composition was affected by lithium, oxidized nitrogen, electrical conductivity, and sulphate levels in the sampled water. We conclude that changes in diatom community composition in the Karoo do reflect the water chemistry and could be useful as bioindicators.