Background The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei. Results In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci. Conclusion With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.