The Cryptococcus species complex consists of two species, Cryptococcus neoformans and Cryptococcus gattii, which cause systemic infections in both immunocompromised and immunocompetent patients. Both species have a bipolar mating system, with mating type (MAT) α being predominant in clinical and environmental isolates. The strains of the Cryptococcus species complex have been divided into eight major molecular types, which show differences in epidemiology, biology and pathogenicity. In this study, two PCR-RFLP analyses, based on the CAP1 and GEF1 genes, which are both located at the MAT locus, were developed for simultaneous identification of the molecular and mating types of isolates of the Cryptococcus species complex. The molecular and mating types of all 144 cryptococcal isolates, including rare subtypes, were successfully determined by both PCR-RFLP approaches. Pattern analysis of the AD hybrids revealed that the serotype A MATa allele in strains of AaDα derived from genotype VNB, whereas the serotype A MATα allele among strains of AαDa and AαDα derived from molecular type VNI.