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The fungal genus Trichoderma (Ascomycota) has a cosmopolitan distribution throughout all climate zones and represents an important factor in soil microbiota. The agricultural importance of these fungi, especially in organic farming, lies in their effective antagonism against soil-borne plant pathogens and the ability to promote plant growth. They are currently marketed as active ingredients of biopesticides (fungicides, nematicides) and separately as plant growth enhancers and bio-stimulants of natural disease resistance in plants. The genus is hyperdiverse at the species level and it is impossible to accurately identify species solely based on their morphology due to the immense similarity of morphological characters in closely related taxa and an increasing number of newly described species which has led to numerous misidentifications or indeterminacies. Regarding the ecological and agricultural importance of Trichoderma species, it is first important to understand their biodiversity and biogeography, while correct species identification is a prerequisite for the registration of adequate Trichoderma-based pesticides. The biodiversity and biogeographical patterns of the genus Trichoderma on the Croatian territory are poorly known because previous studies were scarce. In order to assess the genetic diversity and occurrence of Trichoderma species in the Mediterranean region of Croatia, a total of 50 Trichoderma strains were isolated from the rhizosphere of agricultural plants, bulk soil and dead wood. Trichoderma species were identified on the basis of molecular sequence data (ITS, rpb2, and tef1 gene markers) in combination with the morphological characters and their evolutionary relationships were presented in the phylogenetic tree.