ABSTRACT Hemiascomycetes are species of yeasts within the order Saccharomycetales. The order encompasses disparate genera with a variety of life styles, including opportunistic human pathogens (e.g., Candida albicans ), plant pathogens (e.g., Eremothecium gossypii ), and cosmopolitan yeasts associated with water and decaying vegetation. To analyze the phylogeny of medically important species of yeasts, we selected 38 human pathogenic and related strains in the order Saccharomycetales. The DNA sequences of six nuclear genes were analyzed by maximum likelihood and Bayesian phylogenetic methods. The maximum likelihood analysis of the combined data for all six genes resolved three major lineages with significant support according to Bayesian posterior probability. One clade was mostly comprised of pathogenic species of Candida . Another major group contained members of the family Metschnikowiaceae as a monophyletic group, three species of Debaryomyces , and strains of Candida guilliermondii . The third clade consisted exclusively of species of the family Saccharomycetaceae. Analysis of the evolution of key characters indicated that both codon reassignment and coenzyme Q 9 likely had single origins with multiple losses. Tests of correlated character evolution revealed that these two traits evolved independently.