The molecular diversity of Diaporthe helianthi (anamorph Phomopsis helianthi), the causal agent of sunflower stem canker, was studied in 16 isolates of different geographic origin using nuclear and mitochondrial markers. PCR products corresponding to the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal RNA gene, and to the mitochondrial atp6 gene were sequenced. The ITS1 and ITS2 sequences were compared with those of Phomopsis spp. and Diaporthe spp. obtained from databases. The diversity in the region surrounding the atp6 gene was also studied by restriction analysis using four enzymes. The analyses revealed a marked diversity within the sunflower-isolated strains, which appear to belong to phylogenetically unrelated groups. Noticeably, all the isolates collected in France and in the former Yugoslavia, where severe epiphytotics of sunflower stem canker are frequently reported, showed high similarity to each other forming a clade which clearly differentiated from all other ones within the genus Phomopsis. Conversely, all the isolates collected in Italy, where, despite favourable environmental conditions, the incidence of the disease is low, were only distantly related to the former group and showed sequence similarity with other previously established phylogenetic clades within the Phomopsis/Diaporthe complex.