Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.