Abstract Fungal species from extreme environments represent an underexploited source of stress-resistance genes. These genes have the potential to improve stress tolerance of economically important microorganisms and crops. An efficient high-throughput method for the identification of biotechnologically interesting genes of extremotolerant fungi was developed by constructing a cDNA expression library in Saccharomyces cerevisiae and screening for gain-of-function transformants under stress conditions. The advantages and possible modifications of this method are discussed, and its efficiency is demonstrated using the stress-tolerant basidiomycetous yeast Rhodotorula mucilaginosa. Twelve R. mucilaginosa genes are described that increase halotolerance in S. cerevisiae. These include genes encoding a phosphoglucomutase and a phosphomannomutase. All 12 investigated genes might be useful for the improvement of halotolerance in genetically modified crops or industrial microorganisms.