Twenty-seven isolates of Fusarium oxysporum f. sp. eustomae obtained from diseased Eustoma grandiflorum (lisianthus) plants in north Italy, showing typical fusarium wilt symptoms, were studied for genetic diversity, pathogenicity, and vegetative compatibility (VC). The strains were examined for differences in the nucleotide sequence of translation elongation factor 1-alpha (tef) and two endopolygalacturonase genes (pg1 and pg5). The phylogenetic analysis performed on the isolates enabled the identification of three main groups, named I, II, and III, and two subgroups (Ia and Ib). In pg1 and pg5 sequences, most of nucleotide variations resulted in aminoacid variations, while in tef most of SNPs are in the introns or are silent. By considering the virulence of the strains included in Group I, a low virulence could be observed, Group II and III showed a reduced virulence at 30 A degrees C compared to 20 A degrees C. Some cultivars showed higher tolerance at 30 A degrees C. Principal component analysis (PCA) performed on pathogenicity values, confirmed the three molecular clusters. VC identified two VCGs (vegetative compatibility groups) in Group II, one VCG in Group III, and vegetatively incompatible isolates grouped together in Group I. The integrated analysis confirmed the presence of virulence variation and genetic diversity among the F. oxysporum f. sp. eustomae isolates studied.