Clinically relevant yeasts are conventionally identified by a combination of phenotypic tests, which occasionally provide ambiguous results for atypical isolates or uncommon species. In this study, we evaluate a direct polymerase chain reaction-sequencing method, which exploits sequence divergence in the hypervariable D2 region of the large subunit of the 25-28S ribosomal RNA (rRNA) gene for identification of facultative pathogenic asco- and basidiomycota. A panel of 53 yeasts, including 40 clinical isolates and 13 reference strains representative of some clinically relevant taxa, was investigated by combining standard phenotypic tests with commercial identification systems (RapID, API 20C AUX), and results were compared with the taxonomic allocations inferred by D2 sequence analysis. Species-level resolution was achieved for almost all (52/53) strains by combining internet-based D2 sequence homology (BLAST and FASTA) searches in free-access synchronised databases with phylogenetic analysis. The phylogenetic information carried by the short D2 sequence substantiates a pattern of molecular evolution, which is similar to that inferred from analysis of the larger D1/D2 region, and consistent with previously published 25-28S rRNA phylogenetic architectures of facultative pathogenic yeast, including recently identified species. Inconsistency between conventional and molecular identification results was observed for 11/53 strains, likely on account of the ambiguous interpretation of phenotypic tests.