Genome-wide mutagenesis is a powerful method for identifying new genes that contribute to a phenotype of interest. For many fungal pathogens of plants and animals, Agrobacterium tumefaciens-mediated transformation (ATMT) serves as an efficient insertional mutagen. In Histoplasma capsulatum, the T-DNA element transferred by Agrobacterium stably integrates into the genome, and the majority of mutants contain single copies of the inserted sequence. The T-DNA sequence facilitates the determination of the genomic sequence flanking the insertion through hemi-specific PCR techniques, plasmid rescue, or inverse PCR. We present optimized procedures for generating insertional mutants in H. capsulatum using Agrobacterium-mediated transformation and using this for forward and reverse genetic approaches.