21 strains with all typical morphological characteristics of eight Verticillium species (Phyllachorales) were studied in this work, together with representatives from four Hypocreales species (11 strains), that were previously classified as members of the genus. The PCR products from two nuclear genes, i.e. the ITS1-5.8S-ITS2 region and RNA polymerase II largest subunit gene (rpb1), together with four mitochondrial genes, i.e. the small ribosomal rRNA subunit (rns), the two NADH dehydrogenase subunit genes (nad1 and nad3), and the cytochrome oxidase subunit III gene (cox3) were sequenced and analyzed. Similarly, antibodies raised against one strain of each of the species examined (V. nubilum and V. theobromae excluded) were used against the proteins of all other strains. The number and relative area of precipitates formed after crossed electrophoreses served to estimate the degree of immunochemical relatedness. Combined molecular and immunochemical data clarified the phylogenetic relationships of all true Verticillium species and provided a convincing insight into the evolutionary relation of the sect. Nigrescentia with members of the sect. Verticillium and sect. Prostrata that until recently were included in Verticillium.