Sugar beet in Europe is commonly grown in wheat and maize crop rotations and subsequently pile-stored for several weeks. Beet is threatened by the colonization of saprophytic as well as pathogenic Fusarium spp. A tool for reliable identification based on sequence information of the translation elongation factor 1α (TEF-1α) gene was developed for the numerous Fusarium spp. being isolated from sugar beets. In all, 65 isolates from different species (Fusarium avenaceum, F. cerealis, F. culmorum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. redolens, F. solani, F. tricinctum, and F. venenatum) were obtained from sugar beet at different developmental stages from locations worldwide. Database sequences for additional species (F. sporotrichioides, F. poae, F. torulosum, F. hostae, F. sambucinum, F. subglutinans, and F. verticillioides), isolated from sugar beets in previous studies, were included in the analysis. Molecular sequence analysis of the partial TEF-1α gene fragment revealed sufficient variability to differentiate between the Fusarium spp., resulting in species-dependent separation of the isolates analyzed. This interspecific divergence could be translated into a polymerase chain reaction restriction fragment length polymorphism assay using only two subsequent restriction digests for the differentiation of 17 of 18 species.