Abstract The hypersaline Inland Sea in Qatar constitutes a unique ecosystem characterized by salinities up to saturation, extreme temperature fluctuations, and limited rainfall. To reveal the fungal diversity of this environment, we isolated fungi from water samples collected at the Inland Sea. Taxonomic identification of the isolates was done via DNA barcoding of the ITS1 and ITS2 ribosomal DNA (rDNA) domains and the D1/D2 domains of the nuclear large subunit rDNA. Additional genes, including glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor 1-alpha (tef1), were included for isolates of Alternaria, actin (Act) for Cladosporium, part of the beta-tubulin (BenA) and calmodulin (CaM) genes for Aspergillus and Penicillium. In total, 159 fungal isolates, including 85 Ascomycota and 74 Basidiomycota, were obtained from the water samples collected during four samplings in the winter and summer seasons. About 14% (22/159) of the strains, presumably novel species, were preliminarily identified to the genus level. This is the first report highlighting the diversity of fungi from the hypersaline Inland Sea in Qatar.