AbstractDimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA‐guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9‐expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium‐mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene‐editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectorsSupport Protocol 1: Choosing protospacers in the target geneBasic Protocol 2: Agrobacterium‐mediated transformation of BlastomycesSupport Protocol 2: Preparation of electrocompetent AgrobacteriumSupport Protocol 3: Preparation and recovery of Blastomyces frozen stocks