The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC(90) (0.125 mug/ml) for all C. gattii isolates, followed by a 1 log(2) dilution step increase (MIC(90), 0.25 mug/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC(90) of 0.5 mug/ml, but fluconazole and flucytosine had relatively high MIC(90)s of 8 mug/ml