Pyricularia oryzae is a plant pathogenic fungus that severely affects rice production. Past studies, primarily using mutants generated by spontaneous mutations or artificial physical and chemical mutagenesis, have determined that melanin is required for appressorium turgor, penetration, and virulence of P. oryzae. However, these roles need to be verified by gene knockout and/or overexpression in different strains considering the potential differences in the level of virulence. Here, we confirmed the indispensable roles of melanin in the development and virulence of P. oryzae by knocking out and over-expressing three melanin synthesis genes (ALB1, RSY1, and BUF1) in two wild-type strains (Guy11 and 70-15). Deletion of ALB1, RSY1, or BUF1 led to loss of melanin and virulence in both strains. ALB1, RSY1 and BUF1 in Guy11, and BUF1 in 70-15 were required for conidiation, respectively. ALB1, RSY1, and BUF1 were required for conidial resistance to environmental stresses (UV exposure, oxidization, and freezing damage) in both strains. Guy11 cells had greater amounts of melanin and more transcripts of melanin synthesis genes than 70-15 cells. Paired culture experiments between the deletion or over-expression mutants of melanin synthesis genes suggested that the reaction catalyzed by Buf1, but not Alb1 and Rsy1, was likely a rate-limiting step in melanin biosynthesis in 70-15. These results expand our understanding on melanin and its synthesis genes in P. oryzae as well as its responses to biotic and abiotic environments.