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Persistent cold temperatures, a paucity of nutrients, freeze-thaw cycles, and the strongly seasonal light regime make Antarctica one of Earth’s least hospitable surface environments for complex life. Cyanobacteria, however, are well-adapted to such conditions and are often the dominant primary producers in Antarctic inland aquatic environments. In particular, the network of meltwater ponds on the ‘dirty ice’ of the McMurdo Ice Shelf is an ecosystem with extensive cyanobacteria-dominated microbial mat accumulations. Intact polar lipids (IPLs), heterocyte glycolipids (HGs), bacteriohopanepolyols (BHPs) and sterols in combination with 16S and 18S rRNA gene sequences shed light on the microbial diversity of ponds in this unique polar ecosystem. The high abundance of polyunsaturated IPLs in the Antarctic microbial mats suggests that these lipids provide an important mechanism to maintain membrane fluidity in polar environments. High abundances of HG keto-ols and HG keto-diols, produced by heterocystous cyanobacteria, further support these findings and reveal a unique distribution compared to those from warmer climates. A diversity of sterols and 18S rRNA data confirm the diversity of the protists and meiofauna in these systems and provide a means to shed light on how such communities could not only have survived, but thrived, in similar refugia during Snowball Earth episodes, that is, times in the past when the Earth was totally encased in ice.