We have determined the nucleotide sequences of about 55% of the region of the DNA topoisomerase II gene (approximately 2.3 kb) isolated from the pathogenic Candida species, C. dubliniensis, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. guilliermondii and C. lusitaniae. Evolutionary relationships among nine Candida species including those of C. albicans and C. glabrata were studied based on the DNA topoisomerase II gene. The nucleotide sequences of 2192 bp, which covered two catalytic domains, ATPase and cutting/resealing, were subjected to phylogenetic analysis. Sequence comparison and evolutionary analysis have revealed that the Candida species tested here are not monophyletic, and the two strains within the species C. tropicalis and C. parapsilosis are too diverse to be in a single species. A wide variety of divergence was observed among the functional domains of DNA topoisomerase II, suggesting that Candida species were in different evolutionary paths at least as regarding the DNA topoisomerase II gene. Sequence information and the observation on the species-specific manner of molecular evolution of DNA topoisomerase II in Candida will be applied to develop a method of identification and characterization of the Candida species in both natural and clinical isolates.