Analysis of ribosomal DNA (rDNA) restriction fragment-length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) was used to investigate the genetic variability and biogeographic distribution of clinical and environmental strains of Cryptococcus neoformans isolated from a limited area of southern Italy, where the selection of a predominant cryptococcal genotype could be expected. All isolates belonged to the species Cr. neoformans variety neoformans serotype A. RFLP analysis of a specific rDNA fragment allowed the distinction of strains of Cr. neoformans from closely related fungal reference species, but neither intraspecies nor intravarieties polymorphism was detected. On the contrary, RAPD fingerprints produced by priming with four different primers [(GTG)5, (GACA)4, M13 core sequence and the 8-mer oligonucleotide (GCGGACGG)] were able to characterize the isolates up to the individual level, indicating the presence of marked heterogeneity among Cr. neoformans serotype A strains in southern Italy.