In this study, we aimed at exploring the diversity and community composition of fungi in four pristine and permanently ice-covered lakes of the McMurdo Dry Valleys, Southern Victoria Land, Antarctica. This offers the unique opportunity to study aquatic fungal communities in extreme environments that have not experienced major alterations (including anthropogenic influence) for thousands of years. For this, we collected several samples at multiple depths from four lakes: Lake Fryxell, Lake Hoare, Lake Miers, and the East and West Lobes of Lake Bonney, during the austral summer 2011-2012. Using a culture-independent approach, based on high throughput sequencing of the small subunit of the ribosomal RNA, we characterized the fungal populations and their relationships with other detected eukaryotes according to major variables such as lake type, depth layer, and habitat type (i.e. brackish vs. freshwater).