The purpose of this study was to investigate the ability of ascomycetous yeasts to assimilate/ferment d-fructose. This ability of the vast majority of yeasts has long been neglected since the standardization of the methodology around 1950, wherein fructose was excluded from the standard set of physiological properties for characterizing yeast species, despite the ubiquitous presence of fructose in the natural environment. In this study, we examined 388 strains of yeast, mainly belonging to the Saccharomycetes (Saccharomycotina, Ascomycota), to determine whether they can assimilate/ferment d-fructose. Conventional methods, using liquid medium containing yeast nitrogen base +0.5% (w/v) of d-fructose solution for assimilation and yeast extract-peptone +2% (w/v) fructose solution with an inverted Durham tube for fermentation, were used. All strains examined (n = 388, 100%) assimilated d-fructose, whereas 302 (77.8%) of them fermented d-fructose. In addition, almost all strains capable of fermenting d-glucose could also ferment d-fructose. These results strongly suggest that the ability to assimilate/ferment d-fructose is a universal phenotype among yeasts in the Saccharomycetes. Furthermore, the fructophilic behavior of Ambrosiozyma platypodis JCM 1843 and Cyberlindnera americana JCM 3592 was characterized by sugar consumption profiles during fermentation.