Colletotrichum graminicola is a filamentous, primarily clonally-reproducing fungus inhabiting a wide range of grasses and cereals. During the past several years, this fungus has emerged as one of the most important pathogens in turfgrass systems. Control of the disease is often unpredictable, possibly due to an imperfect understanding of population structure and pathogen variability. Using Bayesian likelihood analyses of nucleotide sequences from three unlinked nuclear loci, we provide compelling evidence of sympatric, species-level phylogenetic divergence between C. graminicola pathogenic to Zea mays and Colletotrichum specimens causing disease in grasses of the subfamily Pooideae. These data also revealed two lineages emerging along distinct evolutionary trajectories among isolates sampled from Pooideae grasses, including several Pooid species cultivated as turfgrasses. Transposon distribution patterns and RFLP fingerprint analysis extend the phylogenetic estimations, independently supporting the conclusion that several closely related cryptic species of Colletotrichum inhabit the grasses of North America.