Rock varnish is a very slow-growing nanostratigraphic coating consisting of approximately 70% fine-grained clay and 30% iron and manganese oxides that forms on the surfaces of rocks in arid and semiarid climates. The microbial diversity associated with rock varnish collected from the hyperarid Yungay region of the Atacama Desert was investigated using culture-independent biomolecular methods and an adenosine triphosphate (ATP) assay. The extraction of DNA from rock varnish collected at Yungay, a region in which little to no DNA has been extracted from the surface soil (<1 cm) to date, indicates that rock varnish may provide a niche habitat for microbial life where water is essentially absent. The clone library constructed suggests the presence of numerous phylogenetically distinct microorganisms, ranging in diversity from Cyanobacterial to á-proteobacteria lineages. The findings also show that only a few micrometers of varnish material are enough to shelter microbes like Chroococcidiopsis spp. from the intense ultraviolet radiation present in the Atacama Desert. Whether or not microorganisms are involved in its nucleation and/or growth, rock varnish appears to provide a microhabitat resembling cryptoendolithic communities seen on a larger scale.