Abstract We applied multilocus sequence typing (MLST) to investigate the population structure and mode of reproduction of Cryptococcus neoformans var. grubii (serotype A). This MLST system utilizes 12 unlinked polymorphic loci, which are dispersed on nine different chromosomes, and allows the unambiguous identification of closely related strains of serotype A. We compared MLST analyses with the conventional genotyping method of detecting amplified fragment length polymorphisms (AFLPs), and there was excellent correlation between the MLST and AFLP results. However, MLST differentiated a larger number of strains. We analyzed a global collection of isolates of serotype A using both methods, and the results identified at least three genetically distinct subpopulations, designated groups VNI, VNII, and VNB. Groups VNI and VNII are widespread, dominated by isolates with the MATα mating type, and predominantly clonal. Conversely, isolates of group VNB are unique to Botswana, include a significant proportion of fertile strains with the MATa mating type, and manifest compelling evidence of recombination. We have AFLP genotyped >1000 strains of serotype A from different parts of the world, including isolates from several African countries, and, to date, haploid serotype A isolates of group VNB have been found only in Botswana.