A large number of isolates of Phomopsis sp. have been collected from the weed Carthamus lanatus (saffron thistle) in Australia, and their potential as biological control agents for weeds of the Asteraceae has been demonstrated. An analysis of their genetic diversity and a multigene phylogenetic analysis were undertaken to ascertain whether these isolates were distinct from other species of Phomopsis that commonly attack crop species in Australia. Minimal variation was found between the Phomopsis spp. isolated from saffron thistle, except two isolates that appeared to share identity with Diaporthe helianthii and P. viticola. Analysis of the selected isolates from saffron thistle with the nucleotide sequence of the partial ITS and tefl-alpha regions demonstrated that the sequences were distinct from all other species of Phomopsis so far described from crops in Australia. These findings provide strong support for the recognition of these isolates as a separate species of Phomopsis. The implications of these findings are discussed in relation to biological control of saffron thistle.