Knowledge on mutation processes is central to interpreting genetic analysis data as well as understanding the underlying nature of almost all evolutionary phenomena. However, studies on genome-wide mutational spectrum and dynamics in fungal pathogens are scarce, hindering our understanding of their evolution and biology. Here, we explored changes in the phenotypes and genome sequences of the rice blast fungus Magnaporthe oryzae during the forced in vitro evolution by weekly transfer of cultures on artificial media. Through combination of experimental evolution with high throughput sequencing technology, we found that mutations accumulate rapidly prior to visible phenotypic changes and that both genetic drift and selection seem to contribute to shaping mutational landscape, suggesting the buffering capacity of fungal genome against mutations. Inference of mutational effects on phenotypes through the use of T-DNA insertion mutants suggested that at least some of the DNA sequence mutations are likely associated with the observed phenotypic changes. Furthermore, our data suggest oxidative damages and UV as major sources of mutation during subcultures. Taken together, our work revealed important properties of original source of variation in the genome of the rice blast fungus. We believe that these results provide not only insights into stability of pathogenicity and genome evolution in plant pathogenic fungi but also a model in which evolution of fungal pathogens in natura can be comparatively investigated.