Universally primed PCR (UP-PCR) fingerprinting combined with UP-PCR product cross hybridization, and ITS1 ribotyping were used to study the genetic relatedness of strains of Trichoderma and Gliocladium for two purposes: (1) to evaluate the ability of the methods to discriminate closely related strains and as tools to group strains which is necessary to facilitate; (2) identification of markers for development of specific detection assays for selected strains. Included among the strains were one T. harzianum, two T. virens, and one G. roseum that had been selected previously for their antagonistic ability against soil-borne phytopathogens. Similarity among strains, found by cross dot blot hybridization using UP-PCR amplification products, was used to group them into 15 genetic entities. ITS1 ribotyping of the strains was performed by digestion of the PCR amplified rDNA spacer region and electrophoresis of the products. The differences obtained from ribotyping as well as the differences in mobility of the intact spacer region were used for grouping of the strains. The UP-PCR hybridization groups and the ITS1 based groups proved to be consistent, but the resolution of the UP-PCR based approach was superior. The results demonstrate that the combination of UP-PCR and ribotyping can aid in clarifying species distinction in Trichoderma and Gliocladium and has the potential to become a valuable tool for studies of diversity and genetic structure of populations of these fungi. Furthermore, identification of single strains by the specific UP-PCR fingerprint seems feasible.