The thermophilic fungusChaetomium thermophilumhas been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeastSaccharomyces cerevisiae. Hence, we aimed to find genes fromC. thermophilumthat are expressed under the control of different sugars and examine their associated 5’ untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters inC. thermophilum,we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed theC. thermophilumorthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of theXDHpromoter, which allowed us to induce a nuclear export defect on the pre-60S subunit whenC. thermophilumcells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters inC. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12896-023-00791-9.