The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.