The McMurdo Dry Valleys region of eastern Antarctica is a cold desert that presents extreme challenges to life. Hypolithic microbial colonisation of the subsoil surfaces of translucent quartz rocks represent a significant source of terrestrial biomass and productivity in this region. Previous studies have described hypoliths as dominated by cyanobacteria. However, hypoliths that occur in the lower Dry Valleys such as the Miers, Garwood and Marshall Valleys are unusual as they are not necessarily cyanobacteria-dominated. These hypoliths support significant eukaryal colonisation by fungi and mosses in addition to cyanobacteria- dominated bacterial assemblages and so have considerable ecological value in this barren landscape. Here, we characterise these novel hypoliths by analysis of environmental rRNA gene sequences. The hypolithic community was demonstrated to be distinct from the surrounding soil and non-translucent rocks. Hypoliths supported cyanobacterial signatures from the Oscillatoriales and Nostocales. Other heterotrophic bacterial signatures were also recovered, and these were phylogenetically diverse and spanned 8 other bacterial phyla. Archaeal phylotypes recovered were phylogenetically aYliated with the large group of unclassified, uncultured Crenarcheota. Eukaryal phylotypes indicated that free-living ascomycetous fungi, chlorophytes and mosses (Bryum sp.) were all supported by these hypoliths, and these are thought to be responsible for the extensive eukaryotic biomass that develops around quartz rocks.