Recent Cryptococcus gattii infections in humans and animals, including several outbreaks in goats, were the basis of this environmental survey in six provinces of Spain. A total of 479 samples from 20 tree species were studied. Cryptococcus gattii was found for the first time in autochthonous Mediterranean trees in Spain. Fourteen isolates of this pathogen were obtained from seven trees of three different species: 12 from carob (Ceratonia siliqua), one from Mediterranean stone pine (Pinus halepensis) and another from eucalyptus (Eucalyptus camaldulensis). All C. gattii isolates were genotype AFLP4/VGI and mating type alpha, and were found to be genetically identical with C. gattii strains isolated from humans and animals in Spain. This supports the hypothesis that these trees may be a natural source for infection of humans and mammals in the Mediterranean area.