The taxonomic relationship between two microcyclic rust species, Puccinia melampodii and Puccinia xanthii, recorded from a number of Asteraceae hosts, was explored by comparing teliospore morphology, host specificity and sequence data. Teliospore morphology varied between and within individual rust accessions but, in general, teliospores of P. xanthii were larger than those of P. melampodii. Field observations and cross-inoculation greenhouse studies showed that P. melampodii accessions were highly specific to their original host species and unable to establish compatible interactions with related Asteraceae species. Though small amounts of genetic variation were detected between P. melampodii and P. xanthii accessions, the ITS and TEF phylogenetic analyses comprising several other rust species grouped these accessions within a well supported clade. Our data indicate that despite differences between P. xanthii and P. melampodii accessions these rusts cannot readily be separated at the species level, supporting earlier taxonomic conclusions and the recent proposal to group them into a morphospecies. Based on host-specificity data, the name Puccinia xanthii var. parthenii-hysterophorae is proposed to accommodate all Mesoamerican records of P. melampodii associated with the host Parthenium hysterophorus.