The present study investigates further the population structure of Candida dubliniensis and its ability to exhibit microevolution. Using 98 isolates (including 80 oral isolates) from 94 patients in 15 countries, we confirmed the existence of two distinct populations within the species C. dubliniensis, designated Cd25 group I and Cd25 group II, respectively, on the basis of DNA fingerprints generated with the C. dubliniensis-specific probe Cd25. The majority of Cd25 group I isolates (48 of 71, 67.6%) were from human immunodeficiency virus (HIV)-infected individuals, whereas the majority of Cd25 group II isolates (19 of 27, 70.4%) were from HIV-negative individuals (P < or = 0.001). Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions of the rRNA genes from 19 representative isolates revealed the presence of four separate genotypes. All of the Cd25 group I isolates tested belonged to genotype 1, while the Cd25 group II population was comprised of three distinct genotypes (genotypes 2 to 4), which corresponded to distinct clades within the Cd25 group II population. These findings were confirmed using genotype-specific PCR primers with 70 isolates. We also showed that C. dubliniensis can exhibit microevolution in vivo and in vitro as occurs in other yeast species. DNA fingerprinting using the C. dubliniensis probes Cd25, Cd24, and Cd1 and karyotype analysis of multiple oral isolates recovered from the same specimen from each of eight separate patients revealed microevolution in six of eight of the clonal populations. Similarly, sequential clonal isolates from various anatomical sites in two separate patients exhibited microevolution. Microevolution was also shown to occur when two clinical isolates susceptible to fluconazole were exposed to the drug in vitro. The epidemiological significance of the four C. dubliniensis genotypes and the ability of C. dubliniensis to undergo microevolution has yet to be established.