Barnyard grass is one of the most serious rice weeds, often growing near paddy fields and therefore potentially serving as a bridging host for the rice blast fungus. In this study, we isolated three fungal strains from diseased barnyard grass leaves in a rice field. Using a pathogenicity assay, we confirmed that they were capable of causing blast symptoms on barnyard grass and rice leaves to various extents. Based on morphology characterization and genome sequence analyses, we confirmed that these three strains were (SCAU-1), (SCAU-2), and (SCAU-6). The established Avirulence () genes , , and were detected by PCR amplification in SCAU-2, but not in SCAU-1 or SCAU-6. Furthermore, the whole-genome sequence analysis helped to reveal the genetic variations and potential virulence factors relating to the host specificity of these three fungal pathogens. Based on the evolutionary analysis of single-copy orthologous proteins, we found that the genes encoding glycoside hydrolases, carbohydrate esterases, oxidoreductase, and multidrug transporters in SCAU-1 and SCAU-6 were expanded, while expansion in SCAU-2 was mainly related to carbohydrate esterases. In summary, our study provides clues to understand the pathogenic mechanisms of fungal isolates from barnyard grass with the potential to cause rice blast.