Species of macrocyclic, heteroecious grass rusts often have been defined with wide host ranges and variation in spore morphology. Consequently, some are species complexes and contain genetically distinct forms. Molecular analyses, together with morphological and biological methods, provide powerful means to dissect these complexes. Puccinia coronata is a complex species that has a broad telial host range including more than 45 genera of grasses and a narrow aecial host range. Phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS) DNA sequences from 15 aecial and telial collections grouped P. coronata into six distinct clades supporting separation of this complex into four distinct species. Puccinia andropogonis, a common rust of tall prairie grasses in North America, is also a complex species. However, in contrast to P. coronata, P. andropogonis has a narrow telial host range and a broad aecial host range. DNA sequence analysis grouped 15 collections of P. andropogonis into six distinct clades representing at least four distinct species. Speciation of P. coronata appears to have occurred primarily by radiation onto new telial hosts, whereas in P. andropogonis speciation appears to have occurred primarily by radiation onto new aecial hosts.