Accurate, rapid and economical fungal species identification has been a major aim in the mycology field. In this study, our goal was to examine the feasibility of a high resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of 8 clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guiilermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons’ sequencing and agarose gel electrophoresis analysis. Real time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in four hours and without the need for any post amplification handling, and thus, reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological setting.This article is protected by copyright. All rights reserved.