Aspergillus fumigatusis a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treatA. fumigatusinfections, but resistance is increasing due to mutations in genes such ascyp51A,hmg1and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance inA. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations incyp51A,cyp51Bandhmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations inA. fumigatus.