Colletotrichum is a cosmopolitan, anamorphic fungal genus responsible for anthracnose disease in hundreds of plant species worldwide, including members of the Poaceae. Anthracnose disease of the widely planted, non-native, warm-season lawn grass, Eremochloae ophiuroides (centipedegrass), is commonly encountered in the southern United States, but the causal agent has never been identified. We use DNA sequence data from modern cultures and archival fungarium specimens in this study to determine the identity of the fungus responsible for centipedegrass anthracnose disease and provide experimental confirmation of pathogenicity. C. eremochloae sp. nov., a pathogen of centipedegrass, is proposed based on phylogenetic evidence from four sequence markers (Apn2, Apn2/ Mat1, Sod2, ITS). C. eremochloae isolates from centipedegrass shared common morphology and phenotype with C. sublineola, a destructive pathogen of cultivated sorghum and Johnsongrass weeds (Sorghum halepense, S. vulgaris). Molecular phylogenetic analysis identified C. eremochloae and C. sublineola as closely related sister taxa, but genealogical concordance supported their distinction as unique phylogenetic species. Fixed nucleotide differences between C. eremochloae and C. sublineola were observed from collections of these fungi spanning 105 y, including the 1904 lectotype specimen of C. sublineola. C. eremochloae was identified from a fungarium specimen of centipedegrass intercepted at a USA port from a 1923 Chinese shipment; the multilocus sequence from this specimen was identical to modern samples of the fungus. Thus, it appears that the fungus might have migrated to the USA around the same time that centipedegrass first was introduced to the USA in 1916 from China, where the grass is indigenous. The new species C. eremochloae is described and illustrated, along with a description and discussion of C. sublineola based on the lectotype and newly designated epitype.