Over the past 10 years, rust diseases have become increasingly prevalent on certain cultivars of Kentucky bluegrass. This pattern suggests that new races or new species of rust fungi may have emerged. To test this hypothesis, 66 samples of turfgrass rust fungi collected from across the United States were evaluated based on sequences of the internal transcribed spacer (ITS)-5.8S rDNA region. Phylogenetic analysis revealed three species: Puccinia coronata, P. graminis, and P. striiformis, comprising 67, 28, and 5% of the samples, respectively. P. coronata was frequently found in association with Kentucky bluegrass, a host-pathogen relationship that has not been previously reported. Comparison of molecular analyses with the use of standard field identification techniques-host association and pustule pigmentation-showed that 58% of the Kentucky bluegrass samples would have been incorrectly diagnosed using nonmolecular criteria. To avoid such misidentifications, a real-time polymerase chain reaction diagnostic protocol was developed for turfgrass-associated P. graminis, P. coronata, and P. striiformis using ITS sequences. Accurate, reproducible, species-specific identifications were made using as few as 50 to 150 urediniospores, even in mixed infections. This study represents the first DNA-based evaluation of turfgrass rust fungi and provides a quick and reliable sequence-based protocol as an alternative to less reliable field-based identification techniques.