Abstract: Objective To evaluate the efficiency of PCR-restriction fragment length polymorphism （PCR-RFLP） aiming at the structure gene g6341, versus PCR fingerprinting analysis in the genotyping of Cryptococcus neoformans . Methods Eight reference strains and 68 clinical and environmental isolates of C. neoformans were genotyped by PCR-RFLP and PCR fingerprinting. In PCR fingerprinting, the minisatellite-specific core sequence of wild-type phage M13 was used as a single primer. The structure gene g6341 was selected for PCR-RFLP analysis by sequence alignments of multiple genes, a pair of primers were developed based on the conserved region of g6341 gene. PCR products were digested with the appropriate restriction endonucleases, and RFLP profiles were analyzed. Partial sequence analysis of g6341 gene was performed for different genotypes of C. neoformans . Phylogenetic analysis was done to study the relatedness between these genotypes. Results As sequence homology analysis showed，g6341 gene was suitable for RFLP analysis. In the case of genotyping of 76 C. neoformans strains, the results obtained from PCR-RFLP were consistent with those from PCR fingerprinting. Sequence analysis of g6341 gene revealed a homology of 84%-97％ among the eight genotypes as well as a consistency of 99%-100% within a same genotype. In the phylogenetic tree, genotypes VNⅠ, VNⅡ, VNⅢ and VNⅣ belonged to one cluster, and genotypes VGⅠ, VGⅡ, VGⅢ and VGⅣ to another cluster. Conclusions PCR-RFLP analysis aiming at the structure gene g6341 is a useful tool to genotype C. neoformans . Sequence analysis of g6341 gene can disclose the relatedness among different molecular types of C. neoformans .