Two members of the Cryptococcus neoformans-gattii species complex, the etiologic agents of cryptococcosis, can be differentiated by biological, biochemical, serological and molecular typing techniques. Based on their differences in carbon and nitrogen utilization patterns, cost effective and very specific diagnostic tests using D-proline and canvanine-glycine-bromthymol blue (CGB) media have been formulated and are widely used for identification of the two species. However, these methods have yet to be tested for strains with confirmed molecular types to assess the degree of specificity for each molecular type in the two species. We collected global isolates of every major molecular type available and tested their patterns of nitrogen utilization. We confirmed specificity of the CGB test to be 100% regardless of molecular type while the D-proline test yielded 8-38% false negative results in three of the four C. gattii molecular types, VGI-VGIII. The utilization pattern of a new set of amino acids: D-alanine, L-tryptophan and L-phenylalanine, showed species specificity comparable to that of D-proline. We discovered that the transcription factor Gat1 (Are1) regulates the utilization of nitrogen differently between C. neoformans and C. gattii strains. Unlike in C. neoformans, expression of the genes encoding glycine decarboxylase complex in C. gatti was only partially suppressed by nitrogen catabolite repression in the presence of ammonium. GAT1 in C. neoformans controlled the induction of three of the four genes encoding the glycine decarboxylase complex when glycine was used as the sole nitrogen source while in C. gattii its regulation of these genes was less stringent. Moreover, while virulence of C. neoformans strains in mice was not affected by Gat1, the transcription factor positively influenced the virulence of C. gattii strain.