The genetic affiliation of a large number of isolates of the Cryptococcus neofornqans species complex from environmental sources in Brazil has been investigated using amplified fragment length polymorphism (AFLP). The strains of C. neoformans isolated from a single tree, as well as from neighbouring trees, showed high similarity values ( > 95%) of their AFLP patterns, thus suggesting considerable genetic homogeneity. The majority of isolates of C. neoforrnans belonged to AFLP genotype 1, and had serotype A and mating type a ( = C. neoformans var. grubii). Three isolates belonged to AFLP genotype 2, with serotype D and mating type a ( = C. neofornzans var. neoformans). One isolate, obtained from a building in Rio de Janeiro inhabited by pigeons, belonged to the AD hybrid AFLP genotype 3. All isolates from trees of C. neoforrnans var. gattii ( = C. gattii) belonged to AFLP genotype 6, and their banding patterns showed relatively low genetic homogeneity with a similarity value of about 76%. Isolates of this genotype occupy an environmental niche in the Americas, and they may cause disease in non-AIDS and AIDS patients as well.