AbstractBrines are hypersaline solutions which have been found within the Antarctic permafrost from the Tarn Flat area (Northern Victoria Land). Here, an investigation on the possible presence and diversity of fungal life within those peculiar ecosystems has been carried out for the first time. Brines samples were collected at 4- and 5-meter depths (TF1 and TF2, respectively), from two brines separated by a thin ice layer. The samples were analyzed via Illumina MiSeq targeting the ITS region specific for both yeasts and filamentous fungi. An unexpected high alpha diversity was found. Beta diversity analysis revealed that the two brines were inhabited by two phylogenetically diverse fungal communities (Unifrac value: 0.56, p value < 0.01; Martin’s P-test p-value < 0.001) characterized by several specialist taxa. The most abundant fungal genera were Candida sp., Leucosporidium sp., Naganishia sp. and Sporobolomyces sp. in TF1, and Leucosporidium sp., Malassezia sp., Naganishia sp. and Sporobolomyces sp. in TF2. A few hypotheses on such differentiation have been done: i) the different chemical and physical composition of the brines; ii) the presence in situ of a thin layer of ice, acting as a physical barrier; and iii) the diverse geological origin of the brines.