A new molecular taxonomic method applicable to the identification of medically important Candida species and other yeast species has been developed. It is based on the electrophoretic pattern of total tRNA samples (a ‘tRNA profile’) isolated from Candida species and generated using high‐resolution semi‐denaturing urea‐polyacrylamide gel electrophoresis and methylene blue staining. Species‐specific tRNA profiles for the species. C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, C. glabrata and Pichia guilliermondii were obtained. Detailed studies with the major human pathogen of the Candida genus, C. albicans, demonstrated that the tRNA profile for a given species was both reproducible and strain‐independent; seven different C. albicans strains generated identical tRNA profiles. Minor strain‐specific heterogeneities in the tRNA profiles of C. guilliermondii and C. parapsilosis were detected, but in neither case did they significantly alter the species‐specific diagnostic tRNA profile. The potential of this method in clarifying taxonomic anomalies was demonstrated by the finding that Type I and Type II strains of C. stellatoidea generate very different tRNA profiles, with that of a Type II strain being identical to the C. albicans tRNA profile. This method offers a number of advantages over current electrophoretic karyotype methods for species identification, both within the Candida genus and with yeast species in general.