Rhizoctonia solani AG1IA is a major generalist pathogen that causes sheath blight. Its genome, which was the first to be sequenced from the Rhizoctonia genus, may serve as a model for studying pathogenic mechanisms. To explore the pathogen-host fitness mechanism of sheath-blight fungus, a comprehensive comparative transcriptome ecotype analysis of R. solani AG1IA isolated from rice, soybean and corn during infection was performed. Special characteristics in gene expression, gene ontology terms and expression of pathogenesis-associated genes, including genes encoding secreted proteins, candidate effectors, hydrolases, and proteins involved in secondary metabolite production and the MAPK pathway, were revealed. Furthermore, as an important means of pathogenic modulation, diverse alternative splicing of key pathogenic genes in Rhizoctonia solani AG1IA during infections of the abovementioned hosts was uncovered for the first time. These important findings of key factors in the pathogenicity of R. solani AG1IA ecotypes during infection of various hosts explain host preference and provide novel insights into the pathogenic mechanisms and host-pathogen selection. Furthermore, they provide information on the fitness of Rhizoctonia, a severe pathogen with a wide host range.