The bacterial cluster regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is a powerful genome editing tool due to its simplicity, versatility and broad applicability. To aid in CRISPR/Cas9 genome editing in fungal plant pathogens, we have developed bioinformatics tools and performed genome-wide prediction of highly specific guide RNA (gRNA) spacers in the genomes of important pathogenic fungi. Using Magnaporthe oryzae and Fusarium graminearum as examples, the polycistronic tRNA-gRNA (PTG) strategy was demonstrated for efficient expression of gRNAs and multiplex genome editing based on the endogenous tRNA processing system. Following the induction of double stranded breaks (DSBs) by Cas9, the efficiency of targeted mutagenesis or gene replacement via non-homologous end joining (NHEJ) or homology-directed repair (HDR) was evaluated in the absence or presence of donor DNA templates. High efficiencies of targeted mutagenesis were achieved in M. oryzae and F. graminearum, leading to the generation of single, double and triple gene mutants. The CRISPR/Cas9 tools and genome editing strategies derived from this study are expected to facilitate the functional discovery of fungal genes and the potential application of gene drive for biocontrol of devastating fungal plant diseases.