Cryptococcus neoformans causes the second most common opportunistic infection in patients with AIDS. In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by C. neoformans and all varieties are recognized as etiological agents of cryptococcosis. This pathogen is a ubiquitous environmental yeast, commonly associated with avian excreta and decaying wood, especially Eucalypt species. The aim of the present study was to search for C. neoformans in Eucalypts and analyze the genotypic diversity of the obtained isolates by RAPD and PCR fingerprinting. All obtained isolates have been C. neoformans var. neoformans, serotype D molecular type VNIV. Serotype D, was isolated from 3 (37.5%) out of 8 cities surveyed in the South Brazilian state Rio Grande do Sul. Nine (9%) out of 99 environmental samples were obtained from Eucalypt species, Eucalyptus calmadulensis and Eucalyptus tereticornis. Molecular analysis using RAPD and PCR-fingerprinting revealed very little genetic diversity in the obtained cryptococcal serotype D isolates. To our knowledge this is the first report of the isolation of serotype D from Eucalyptus trees in Brazil. More studies are required in order to establish the ecological significance of this finding.