Abstract: Many Verticillium species are soil-borne fungal pathogens which cause each yearworldwide crop losses in the range of billions of dollars. Rapid and reliable diagnostic methodsare needed to detect and differentiate Verticillium species in soil. Existing cultivation-dependantdiagnostic methods for Verticillium spp. do not clearly discriminate among V. dahliae, V. longisporum,and V. tricorpus while detection of V. albo-atrum is not possible. However, onlyV. dahliae and V. albo-atrum are pathogenic to strawberries. Therefore, reliable methods forspecific detection and differentiation of these two Verticillium species in soil are needed. As afirst step in the development of a specific PCR-based diagnostic tool the molecular geneticphylogeny of Verticillium spp. was analyzed using a multi gene approach. Gene sequences wereall retrieved from GenBank and included sequences for the ribosomal operon, i.e. the smallsubunit ribosomal RNA (SSU rRNA), the internal transcribed spacer (ITS) and the intergenicspacer (IGS), as well as sequences for the DNA-dependent RNA polymerase II largest subunit(rpb1) and the cytochrome oxydase (cox3). Cluster analyses revealed species-dependentclustering for the IGS gene sequences. Therefore, the complete IGS region was isolated from 50Verticillium spp. strains and DNA sequences were determined. Based on this data set, speciesspecificDNA sequence signatures will be identified and primers specifically targeting the IGS ofthe different species will be designed. Specificity of the primers will be tested on the straincollection and on environmental samples before application as a diagnostic tool for detection andquantification of Verticillium wilt infestation in the field.