Anthracnose caused by Colletotrichum sublineola is an important disease of cultivated sorghum (Sorghum bicolor) worldwide. Anthracnose is also common on the ubiquitous wild sorghum relative Johnsongrass (S. halepense). Analysis of repetitive molecular fingerprinting markers revealed that isolates of C. sublineola from both hosts in the southeastern United States were genotypically diverse, with relatively few haplotypes found in more than one location. With few exceptions, isolates recovered from S. bicolor belonged to a population that was genetically distinct from the population recovered from S. halepense. Twenty-three isolates from cultivated sorghum were all pathogenic to at least one of 13 heritage inbred lines of S. bicolor. In all, 4 of 10 isolates from S. halepense were also pathogenic to one or more of the lines, while the rest caused no disease in greenhouse assays. The four pathogenic isolates from S. halepense were less aggressive, on average, than isolates from S. bicolor, although the ranges overlapped. Pathogenicity tests involving 15 representative pathogenic isolates from S. bicolor and S. halepense on eight heritage inbred lines of S. bicolor identified 12 races. The combined results of this study demonstrated that C. sublineola comprises two separate host-associated subpopulations in the field, even though some isolates from S. halepense were able to cause disease on S. bicolor under ideal greenhouse conditions. Nonetheless, the apparent existence of infrequent cross-infection events in the field, indicated by molecular fingerprinting, suggests that Johnsongrass has the potential to serve as a refuge and an incubator for genetic diversity in C. sublineola, which can complicate efforts to develop and deploy resistant sweet sorghum varieties in the region.