Cryptococcus isolates from AIDS patients in southern California were characterized by molecular analyses. Pheromone MFalpha1 and MFa1 gene fragments were polymerase chain reaction-amplified with fluorescently labeled primers and analyzed by capillary electrophoresis (CE) on DNA analyzer. CE-fragment-length analyses (CE-FLAs) and CE-single-strand conformation polymorphisms (CE-SSCPs) were used to determine Cryptococcus gattii (Cg), C. neoformans (Cn) varieties neoformans (CnVN) and grubii (CnVG), mating types, and hybrids. Corroborative tests carried out in parallel included growth on specialized media and serotyping with a commercial kit. All 276 clinical strains tested as haploid MATalpha by CE-FLA. CE-SSCP analyses of MFalpha1 showed 219 (79.3%) CnVG, 23 (8.3%) CnVN, and 34 (12.3%) Cg isolates. CE-FLA and CE-SSCP are promising tools for high-throughput screening of Cryptococcus isolates. The high prevalence of Cg was noteworthy, in view of its sporadic reports from AIDS patients in North America and its recent emergence as a primary pathogen on Vancouver Island, Canada.