With genome sequences of more and more fungi become available, high-throughput systematic -mutagenesis is desirable for functional genomics studies. While a number of random insertional mutagenesis and targeted gene disruption approaches have been used in filamentous fungi, Agrobacterium tumefaciens-mediated Transformation (ATMT) remains one of the most effective methods for identifying genes required for specific fungal developmental or infection processes. Because of its simplicity, ATMT is suitable for large-scale insertion mutagenesis in fungi. Magnaporthe oryzae, the rice blast fungus is a model for studying host-pathogen interactions. Here, we describe protocols for generating a M. oryzae mutant library consisting of over 70,000 ATMT transformants and for identifying genes -disrupted by T-DNA in the mutants by TAIL-PCR.