Agrobacterium tumefaciens- mediated transformation (ATMT) has become an important tool for functional genomics in fungi. ATMT-based approaches such as random insertional mutagenesis and targeted knockout are widely used for gene functional analysis in plant-pathogen interactions. Here, we describe a protocol for the identification of pathogenicity and virulence genes through random insertional mutagenesis using the fungal wilt pathogen Verticillium dahliae as an example for the protocol.