Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyteArthroderma vanbreuseghemii(anamorph:Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeastSaccharomyces cerevisiaeas a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as theflpgene adapted to the pathogenic yeastCandida albicans(caflp) or a synthetic codon-optimizedflp(avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Bothflpgenes were under control of theTrichophyton rubrumcopper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only theavflp-harbouring module was functional inA. vanbreuseghemii. Applying this system, we successfully produced theKu80recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with doubleMEPor tripleSUBgene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.