Fusarium avenaceum is a globally distributed fungus commonly isolated from soil and a wide range of plants. Severe outbreaks of crown and stem rot of the flowering ornamental, lisianthus (Eustoma grandiflorum), have been attributed to F. avenaceum. We sequenced portions of the translation elongation factor 1-alpha (tef) and beta-tubulin (benA) protein coding genes as well as partial intergenic spacer (IGS) regions of the nuclear ribosomal genes in 37 Fusarium isolates obtained from lisianthus and other host plants. Isolates that were previously identified morphologically as F. acuminatum were included as an outgroup. Phylogenetic analyses of tef, benA, and IGS sequences showed that F. avenaceum isolates were an exclusive group with strong bootstrap support and no significant incongruence among gene genealogies. Isolates from lisianthus were scattered within this clade and did not form distinct groups based on host species or locality. Pathogenicity tests of F. avenaceum isolates obtained from several other hosts showed an ability to cause disease on lisianthus, suggesting that F. avenaceum may be pathogenic on lisianthus regardless of its phylogenetic origin. These findings have management implications and suggest that any host that supports F. avenaceum may serve as a source of inoculum for lisianthus growers.